diff --git "a/deduped/dedup_0070.jsonl" "b/deduped/dedup_0070.jsonl" new file mode 100644--- /dev/null +++ "b/deduped/dedup_0070.jsonl" @@ -0,0 +1,48 @@ +{"text": "Peach is being developed as a model organism for Rosaceae, an economically important family that includes fruits and ornamental plants such as apple, pear, strawberry, cherry, almond and rose. The genomics and genetics data of peach can play a significant role in the gene discovery and the genetic understanding of related species. The effective utilization of these peach resources, however, requires the development of an integrated and centralized database with associated analysis tools.The Genome Database for Rosaceae (GDR) is a curated and integrated web-based relational database. GDR contains comprehensive data of the genetically anchored peach physical map, an annotated peach EST database, Rosaceae maps and markers and all publicly available Rosaceae sequences. Annotations of ESTs include contig assembly, putative function, simple sequence repeats, and anchored position to the peach physical map where applicable. Our integrated map viewer provides graphical interface to the genetic, transcriptome and physical mapping information. ESTs, BACs and markers can be queried by various categories and the search result sites are linked to the integrated map viewer or to the WebFPC physical map sites. In addition to browsing and querying the database, users can compare their sequences with the annotated GDR sequences via a dedicated sequence similarity server running either the BLAST or FASTA algorithm. To demonstrate the utility of the integrated and fully annotated database and analysis tools, we describe a case study where we anchored Rosaceae sequences to the peach physical and genetic map by sequence similarity..The GDR has been initiated to meet the major deficiency in Rosaceae genomics and genetics research, namely a centralized web database and bioinformatics tools for data storage, analysis and exchange. GDR can be accessed at Malus), pear (Pyrus), raspberries/blackberries (Rubus), strawberries (Fragaria), and stone fruits (Prunus) such as peach/nectarine, apricot, plum, cherry and almond [Arabidopsis and other characteristics: a relatively short juvenile period (2\u20133 yrs) and extensive genetics and genomics resources such as molecular marker maps, interesting mutants and clone library resources [In the United States and temperate regions throughout the world, the family Rosaceae ranks third in economic importance. The most important fruit producing crops include apple has been initiated. The goals of the GDR are (1) to develop an organized and integrated web resource for peach genomics data to facilitate the gene discovery in other member species by a comparative mapping approach, (2) to collect and integrate all Rosaceae genomics data, (3) to develop online tools and resources for the Rosaceae community. In this paper, we describe the structure and content of the database, we review the database access utility and tools and we report a case study where we mapped publicly available Rosaceae sequences to the peach physical and genetic maps by sequence similarity.The volume and the complexity of the data being produced by these peach genomics projects, in addition to the rapidly accumulating genomics and genetics data for other important rosaceous species, necessitate the development of a properly curated and integrated scientific database. Such a database will help scientists to efficiently access, analyze, integrate and apply the data to their own research in a timely manner. RoseDB, which focused on apple genetics and cherry genomics, has been decommissioned and now only exists as a mirror site at INRA. To meet the major need for a centralized and integrated web-database for genomics and genetics research in Rosaceae, the Genome Database for Rosaceae and the singletons that have either unique protein matches or no known significant matches. Peach ESTs were further annotated by Gene Ontology (GO) assignment based on the single \"best hit\" match against the SWISS-PROT database. Of the 1552 sequences from the putative peach unigene set that had matches with the SWISS-PROT database, 1439 could tentatively be assigned GO classifications. Additional sequence annotation includes computational analysis for simple sequence repeats (SSR) and open reading frame (ORF) on both the filtered clone library and the contig library. SSR analysis was preformed using a modified version (CUGISSR) of a Perl script SSRIT [. FLIP is a UNIX C program that finds/translates ORFs (open reading frames) in sequences. Using the FLIP output, CUGISSR selects the longest ORF as the putative coding region and reports the location of SSRs in relation to the putative coding region.The peach and almond ESTs are being processed at the Clemson University Genomics Institute (CUGI) utilizing publicly available software, integrated in a fully automated in-house developed script (CUGIEST). The processing occurs in three stages: trace file processing to identify a filtered, high quality clone library, assembly of high quality sequences to produce longer transcripts and reduce redundancy, and sequence annotation. Annotation consists of pairwise comparison of both the filtered clone library and the EST contig consensus sequences against the GenBank nr protein database using the fastx3.4 algorithm . The tenpt SSRIT with parpt SSRIT program -9) following a monthly homology search of GenBank nr protein database using the fastx34 algorithm.In addition to the peach and almond ESTs processed by CUGI, all the publicly available Rosaceae EST data are daily downloaded from GenBank dbEST and annotated with the top ten most significant matches . The SVG viewer plug-in can be freely downloaded from the Adobe Website and the system requirements can be found at their website . Our map viewer program accesses our underlying relational database to dynamically generate an integrated genetic and transcript map with a direct link to the WebFPC physical map from each marker.Web interfaces for database query and the query result pages are mostly developed using Java Server Page (JSP). JSPs are more efficient, easier to use, more powerful and more portable than traditional CGI and many alternative CGI . BAC conThe GDR website is composed of general information pages, database query/browse interfaces and other tools such as map viewer and sequence similarity server. The GDR web pages are extensively linked such that users can easily access the data of interest regardless of the navigation starting point. For example, the EST detail pages have links to the BAC detail pages, marker detail pages or map viewer for the ESTs that are anchored to BACs, markers or maps. Similarly, the BAC detail pages have links to EST detail page, marker detail page, WebFPC and map viewer. Users can also access the data detail pages from the map viewer or WebFPC/WebChrom. Instead of displaying the entire EST or BAC data in one page, we used the right hand side navigation bars to help users find specific information easily and quickly. A general GDR navigation tool bar is also included in each page to help provide a more user-friendly interface.The generic search site allows users to select data types such as EST, BAC and Marker, and search by name. Users can also follow the link to perform more detailed search for each data type. In the EST search site, users can search either the CUGI peach EST database or Rosaceae EST database downloaded from GenBank. ESTs can be searched by their name(s) and annotation features such as whether the EST belongs to a contig, is a unigene, is used as a probe and has SSRs, or any combination thereof. The EST details page, instead of displaying all the details in one page, initially displays the clone information and the sequence with a side bar containing links to library detail, assembly/unigene information, sequence homology, SSR information, Map position and anchored BACs. Each page linked from this side bar has the same side bar for easy navigation between the features. The Sequence homology page shows the most significant matches (EXP < 1e-9) in the Genbank nr protein database from the fastx sequence similarity search. The SSR information page shows the sequence along with the computationally derived SSRs to help users in the primer design for SSR marker development. The longest putative open reading frame (ORF) is also marked in color in the sequence along with the SSRs. SSRs in the non-coding region tend to be more polymorphic and those in the coding region tend to be more transferable among species, so the information of SSR position in a gene structure will be useful for marker development. The Map position page allows users to view the ESTs' map position using our Map Viewer. Users can retrieve the anchored BAC clones for the EST of interest and all the other ESTs and markers that hybridized to the same BAC through the anchored BACs page. The assembly/unigene information page displays the assembly results which include the contig name and the unigene clone that best represents the contig. The contig name is linked to a contig page that displays the contig sequence with a side bar containing links to the comprising ESTs, sequence homology and SSR information. As ESTs with no match to the GenBank proteins or with no SSRs can still assemble into contigs with a match or SSRs, users may get further annotation results of their ESTs of interest by visiting the contig detail site. In addition, contigs may have longer sequences surrounding the SSRs, allowing more flexibility in the primer design for marker development.In the BAC search site, users can search BACs by name or by probe specifications used for BAC hybridization. The search results site and the linked sites provide users with all the data about the BAC, such as the BAC contigs that the BAC belongs to, other BACs in the contig, probes that hybridized to the BACs, the detailed data about the probes, and link to the WebFPC physical map. Markers can be searched by name or features such as map name, type, and source organism. Similarly with the BAC search result sites, the maker search results sites leads users to pages with the marker information, anchored BACs, other markers and ESTs hybridized to the same BACs and link to the Map Viewer and the WebFPC physical map.Prunus map and each linkage group has a link to a page where the developing contigs are located by the linkage group. Each contig has a direct link to WebFPC in which the individual BAC clones are displayed and various search parameters. Users can upload batch sequence files and the parsed results from the search are formatted in a spread sheet and sent to users by email. When the query sequence has a match to the annotated GDR sequences, users can retrieve all the information such as putative function, SSRs, and the anchored map positions via a hyperlink in the excel spreadsheet. Our sequence similarity server, specifically designed for Rosaceae researchers, will help users utilize the developing peach resources in the studies of other Rosaceae species. For example, as described in the case study below, sequences derived from other Rosaceae species could be immediately anchored to the various Rosaceae maps and the physical map when the query sequences show significant similarity to the mapped peach ESTs.Prunus sequences such as apricot (Prunus armeniaca), almond (Prunus dulcis) and sour cherry (Prunus cerasus). This was expected as peach also belongs to the genera Prunus. The 259 query/match pairs consisted of 209 Rosaceae sequences and 61 mapped peach ESTs. The matching of multiple Rosaceae sequences to single peach sequences was expected since the Rosaceae sequences were not assembled and therefore potentially contains multiple sequences representing the same gene. The 209 Rosaceae sequences were anchored to 38 different loci in four different Rosaceae maps. The number of sequences from each Rosaceae species that anchored to each map is shown in Figure .We report here a case study illustrating the utility of our sequence similarity server and other integrated GDR web resources. In this study, we performed a sequence similarity search using the FASTA algorithm with the non-peach Rosaceae sequences against the mapped peach ESTs to annotate Rosaceae sequences with map positions. A fasta formatted file with a total of 16258 publicly available non peach Rosaceae sequences was uploaded to the GDR FASTA server. We selected the mapped peach database and used the default parameters. The search results returned from the server are formatted in a spread sheet for easy browsing and the match names are hyperlinked to both GenBank and the GDR web site (Figure Prunus genomics projects. Data to be added in the near future include apple ESTs, strawberry ESTs, rose ESTs and apricot map/marker data from collaborators. When the genomics projects from Prunus are finished, we will host 10\u201315,000 unique ESTs from a variety of vegetative and reproductive tissues of peach and almond, the complete peach physical map with anchored genetic markers and unique ESTs. In addition to adding new data, future development efforts will focus on improving the tools and functionality of the web interface such as an advanced search site with options for search/display categories, full sequence processing facilities for Rosaceae researchers, a newsgroup for the Rosaceae community, a site for Rosaceae literature, and more analysis tools such as an interactive contig viewer and a comparative map viewer.We plan to incorporate more Rosaceae genomics and genetics data from researchers worldwide as well as data from the ongoing The GDR is initiated to support the genomics and genetics research in Rosaceae, which contains numerous economically important fruit trees and horticultural plants. Currently GDR contains all the genomics data for the Rosaceae model peach, maps and markers of Rosaceae species and all the publicly available Rosaceae ESTs. Our integrated database provides users with easy access and retrieval of the annotated data, and the web tools enable them to further analyze their data. With future plans, including more data acquisition and tool developments, GDR will play an important role in the timely and efficient analysis of the data, the exchange of results and ideas among researchers worldwide, the support of Rosaceae labs worldwide with Bioinformatics tools and the utilization of the data from the model species in the study of other Rosaceae species. The methodology and tools applied to develop GDR should be easily applied to develop other comparative genomic databases for different families..The GDR is publicly available and can be accessed at AFLP: Amplified Fragment Length PolymorphismBAC: Bacterial Artificial ChromosomeCGI: Common Gateway InterfaceEST: Expressed Sequence TagEXP: EXpectation ValueHTML: HyperText Markup LanguageHTTP: HyperText Transfer ProtocolINRA: Institut National de la Recherche AgronomiqueRFLP: Restriction Fragment Length PolymorphismSSR: Simple Sequence RepeatXML: eXtensible Markup LanguageSJ performed web interface design and programming for html pages and JSP search pages, participated in the database construction, carried out database curation, designed the Map Viewer and performed the case study. CJ wrote scripts for database upload and sequence processing, programmed Map Viewer and implemented WebFPC and WebChrom. MS participated in the database construction and wrote scripts for data upload and search pages for Genbank Rosaceae sequences. SF performs database administration. IC developed the application prototype for servlet-database connection and participated in the database construction. AA conceived of the project and participated in its design and coordination. ZD developed the sequence similarity server application. JT participated in the coordination of the project. DM conceived and supervised the project, participated in the database construction, carried out EST analysis, and provided input in the web interface design. All authors read and approved the final manuscript."} +{"text": "Evolutionary Genomics and Biodiversity to begin to address this.Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs.EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity. Large-scale genomic technologies have generated an extraordinary amount of data in the past few decades. Consequently, a huge effort has been made toward creating biological databases and systems to organize, analyze, and share information with the world-wide community -4. The aEvolutionary Genomics and Biodiversity (EGenBio) project as a web-based system to simplify large-scale evolutionary data management.Genomic biodiversity has been defined as dense sampling of molecular data from diverse taxonomic groups for large genomic regions or complete genomes , and infEGenBio is to provide integrated analysis and visualization of raw sequence data, alignments, and phylogenetic trees, to rapidly curate and annotate that data, and to filter that data based on these annotations for further analysis of specific genomic contexts. It is designed to be robust to change and easily extensible to other datasets and other analytical programs. To accomplish this, EGenBio has web-based interfaces designed to: (1) access computational tools for phylogenetic and evolutionary analyses; (2) facilitate the construction of large-scale sequence and alignment datasets across diverse taxa; and (3) provide a framework for comparative analysis of diverse genes and genomes. EGenBio may also serve to promote the utility of increases in the scale of genomic biodiversity.The central aim of EGenBio serve to organize web-based access to the primary custom-built tools (Figure Genomics division provides access to pre-aligned sequence databases, and serves as a means of flexibly selecting genes and species/genomes of interest and producing a concatenated and annotated alignment for use in further analysis. The data is stored in a MySQL database and accessed \"on the fly\". Our prototype database contains complete vertebrate mitochondrial genomes that are mostly obtained from NCBI RefSeq annotations [Biodiversity division is a collection of tools for analysis of alignments or sets of sequences (Table Evolution division includes tools (Table The three conceptual divisions of s Figure , Table 1otations , but alsotations , but eitEGenBio contains a Tools section that serves as a repository for small stand-alone tools that may also exist as components of other pages, or which serve other simple purposes. The Help link leads not to a separate section, but rather to what we will call a separate \"framework\". The structure of the Help framework mirrors the main framework exactly, but instead of linking to actual tools, Help pages link to detailed descriptions and documentation for each page. Invisible to most users, a hidden Design framework allows for rapid editing and movement of page and site structure information from design to laboratory testing stages, and finally to public access.In addition to the three main sections, EGenBio is a web-based system for analysis in evolutionary genomics and biodiversity. It provides tools and resources for quickly creating, modifying, and analyzing large alignment datasets in ways that we have found useful in our own computer-based and experimental evolutionary genomics research. Our prototype database of complete vertebrate mitochondrial genomes represents the densest complete set of genes currently available from closely related organisms. It can be accessed flexibly according to comma-separated queries or a phylogenetic tree navigation system. EGenBio is designed to be easily extensible to use with other protein complexes and other analytical programs. Our goal is to incorporate and utilize as many existing programs as possible, and to develop only \"added value\" programs. In the current public version, all tools are novel to our system except that alignments are created using ClustalW [PRAED alignment annotation system based on data filters allows alignments to be modified easily according to user interest in annotation features, and allows for the results of analyses to be returned as further annotations on the alignments. Since it is derived from the NEXUS format, it is easy to add batch commands to direct analyses using many common phylogenetic analysis programs. The PRAED format and data filters are a unique feature of the EGenBio system.ClustalW . The PRAEGenBio include the creation of additional data filters, incorporation of more genes for analysis of functional divergence, development of further visualization tools for statistical analyses of evolutionary dynamics, and automated procedures for analysis using existing programs and tools. We also welcome feedback from the scientific community on areas of general need for integrated evolutionary genomics tools. EGenBio is publicly available and can be accessed at via anonymous login. User accounts that allow users to save search parameters and results are provided upon request. Incorporation and private access to pre-publication data can also be accommodated upon request. Replication of the EGenBio system would require a Linux-based operating system capable of running Perl, Perl-GD, R, PHP, MySQL, and an Apache web server. It would also require installation of numerous custom scripts in addition to ClustalW.Future modules under development in PRAED: PRagmatic Analysis of Evolutionary Data.LRT: Likelihood ratio test; MCMC: Markov chain Monte Carlo; mtDNA: mitochondrial DNA; NCBI: National Center for Biotechnology Information; Help and Design pages and organism database, and co-wrote this manuscript. MTR developed and managed the system and has created numerous tools. ZOW developed one of the tools and assisted in preparation of the manuscript. JJF began initial development of the system and developed several tools. RJ worked on the Help and Design pages, the mirror/framework system, and automated generation of pages. ZJJ developed one of the tools and assisted in preparation of the manuscript. TJM assisted in preparation of the manuscript, and review and editing of the web site, and is developing one of the tools. DDP is the principal investigator responsible for the creation, conceptualization, and management of the EGenBio system, developed early versions of many of the tools, and co-wrote this manuscript.LAN is a scientific database curator responsible for the design and documentation of the"} +{"text": "XGDB, a software infrastructure consisting of integrated tools for the storage, display and analysis of genome features in their genomics context is described. The eXtensible Genome Data Broker (xGDB) provides a software infrastructure consisting of integrated tools for the storage, display, and analysis of genome features in their genomic context. Common features include gene structure annotations, spliced alignments, mapping of repetitive sequence, and microarray probes, but the software supports inclusion of any property that can be associated with a genomic location. The xGDB distribution and user support utilities are available online at the xGDB project website, http://xgdb.sourceforge.net/. The assembled genomic sequence of an organism provides a natural scaffold for organizing biologic data. However, researchers are easily overwhelmed if they do not have the computational tools necessary to interpret the features of these assemblies -4. Altholections -7. The xIt is important to note that xGDB differs from and is complementary to database systems such as GMOD , EnsEMBLad hoc methods for such analyses is expensive and time consuming. This cost is a major deterrent to many research endeavors and often leads to continuous redevelopment of analysis procedures [An extensible infrastructure allows a wide array of data, tools, and analysis results to be brought together and provides the means by which to target their use in a focused manner. The xGDB package has been used to establish unique infrastructures tailored to the evaluation of genomic features. The xGDB instances available at PlantGDB have beeocedures -21. LackArabidopsis thaliana and emerging genomic sequence assemblies of Zea mays, respectively. Additional plant genome xGDB systems are accessible through the PlantGDB website [In the following we first discuss the features and capabilities of an xGDB system as seen by end users. We then present the internal design and back-end components relevant to data providers and private installations. The installation is straightforward and requires basic knowledge of common open source software. For the purposes of illustration, we refer to AtGDB and ZmGD website .The xGDB system is primarily accessed through dynamically generated web pages. These pages can be classified into context, record, and web service pages. Context pages present the location of genomic data sources in relation to surrounding features. Record pages localize pertinent external references, alignment results, and web service links. Web service pages allow a user to interact with data stored in the xGDB system, for example invoking BLAST for sequence comparisons ,25 or GeOn accessing an xGDB system, users are presented with navigational controls that allow them to search for genomic feature records and/or genomic locations. Navigational controls are displayed in a standard header at the top of all pages generated by the xGDB system Figure item 2. Genome context pages contain one or more sources of feature data such as curated gene annotations, locations of genomic markers, alignments of microarray probes, gene structure predictions, and alignments of expressed sequence tags (ESTs), cDNA, or assembled contigs of sequence. Figure Zea mays gene TBP-2 (shown in dark blue) as defined in the GenBank record of BAC accession Z474J15 , or that fails to locate a feature identifier triggers a descriptive search of available feature components. Searches resulting in multiple matching features will display a summary page detailing the matching features and their genomic locations. For example, Figure Record pages provide information and web services pertinent to an individual feature. Users access record pages by clicking on a feature glyph from any context page Figure item 1. A typical record page includes information describing the feature source, peptide/nucleotide sequence(s), alignment coordinates, web service links, pertinent external website links, links to the alignment result on which the feature glyph is based, and tables summarizing the position and quality of the feature aligned to other genomic locations Figure . DisplayA major provision of the xGDB software design is extensibility of the core xGDB infrastructure. As such, extension of xGDB by adding third-party enhancements is encouraged. Two such enhancements, developed concurrently with xGDB, are the yrGATE gene annotation toolkit and the GAEVAL genome annotation evaluation toolkit. Both toolkits include fully functional standalone applications that can be incorporated into xGDB via web service extension modules.The yrGATE toolkit provides an online portal for creation and submission of gene annotation. This web service is suitable for developing a large and nonexclusive community of annotators ranging in experience from professional curator to student. The yrGATE@xGDB extension module provides feature glyphs, search capabilities, context dependent web service links, and connections to evidence features stored in xGDB. This extension allows users to access yrGATE via web service links found on any context page for the purpose of creating an annotation. When xGDB is extended by this module additional navigational links are provided for all xGDB page headers. With these links, user can access the yrGATE annotation management pages that provide user account details, curation tools, and listings of accepted annotations.The GAEVAL toolkit provides a system for the analysis of gene structure annotation by evaluation of supporting and incongruent evidence. This application is suitable for evaluating individual gene annotations by comparing both supporting and incongruent evidence. The GAEVAL@xGDB extension module enhances existing annotation feature components by adding glyph details to each feature, cuing users as to its GAEVAL evaluation. Glyph extensions include flags for exonic sequence coverage, splice site confirmation, and possible instances of alternative splicing, alternative transcriptional termination site usage, annotation fusion, annotation fission, or erroneous annotation overlap Figure item 8. Combining these extensions under the xGDB infrastructure establishes a framework for targeting the efforts of would-be community annotators. Through access to the GAEVAL query service , lists oWe now describe the internal design and back-end components of xGDB accessible to data providers and users desiring private installations. We first present the overall system design, which is focused on modularity and extensibility. We then detail the feature component modules that are distributed with xGDB. Options for integrating alternative database structures and distributed database architecture are then discussed. Finally, we discuss options for installation and custom configuration of an xGDB system.The xGDB system consists of both user interface and data management components. Together, these components make xGDB highly modular and extensible. On the front end, the xGDB user interface is provided by a collection of CGI (common gateway interface) scripts. Core CGI scripts are maintained in data independent modules such that multiple xGDB systems may be operated using a single core installation. The AtGDB and ZmGDB systems illustrated herein, as well as all other species configurations maintained by PlantGDB, operate from a single xGDB core by taking advantage of this design feature. In addition, extended functionality such as that of the GAEVAL@xGDB service can be installed in a centralized location and made optionally accessible to all local xGDB systems.Data management and back-end database interoperability are provided by the xGDB database object and independent feature component modules discussed below. The use of modular feature components allows plug-in like inclusion of new feature sources as well as customization of existing sources. Feature components are built from an object oriented paradigm, in which required methods are gained through object inheritance and can be customized or extended by overriding individual method instances. These methods may take place in either the component class or individual instances of an existing class. Figure Feature component modules consist of a Perl encoded DSO (data source object), web service scripts providing unique functionality to each feature component, data management scripts for loading features from flat files of various formats into a relational database management system, and supporting information necessary for feature configuration and customization. A variety of modules are available in the core xGDB distribution, including those encapsulating GenBank gene features, TIGR transcription units, and GeneSeqer expressed sequence spliced alignments. Incidentally, any genomic feature that can be positioned by a genomic coordinate can be developed into a feature component module. For example, with only minor modification of existing modules, we have added predicted repeats, GSS alignments, and microarray probe positions to the feature component modules in use at PlantGDB. As described in the following text, existing feature component modules and their common DSO design provides an ample infrastructure for managing most genomic features.The DSO of each modular feature component inherits from a rich object framework that allows efficient method inheritance and less coding to develop objects encompassing new genomic feature sources Figure . CurrentMethod callbacks and subroutine hooks are used in the DSO framework to allow single instance customization of often modified object methods such as identifier and descriptive search routines, context region and record link publishers, and feature information HTML generators. The methods inherited from either the Annotation or Sequence objects encode subroutine hooks that allow a DSO to be customized by declaring a 'mod' file as an object configuration parameter. When declared, this 'mod' file is included in the DSO framework for its respective feature component. Although similar in function to Perl modules, a 'mod' file need not adhere to any packaging or naming conventions and is instantiated only when needed by an individual feature. In Figure The xGDB database object manages the individual component features and provides adaptor methods for the relational database system of each component. Using an adaptor methodology, the choice of database management system, host, and scheme can be delegated to each feature component. As such, xGDB is capable of operating under distributed database architectures. One highly appealing use for such architecture is in maintaining an often changing feature set. For instance, local use of the individual EST and cDNA alignment feature available at AtGDB would necessitate a pipeline for continuous update as new sequences become available. This poses a challenge both in resource and time commitment for most small to moderately sized research groups. The ability of xGDB to utilize a distributed architecture, however, allows PlantGDB to provide direct connection to available PlantGDB feature sources Table . TherefoThe variety of genomic features, distribution sources, and distributed formats currently available for genomic context analysis necessitates an infrastructure system with federated data management capabilities. The modular design of xGDB allows creation of feature components specific to any distribution source or format. In addition to its native database architecture, the xGDB system is currently capable of using DAS distribuSetting up an xGDB system requires installation of the core xGDB distribution, installing an xGDB instance, populating a feature component module, and configuring the xGDB instance to include the feature component. Documentation and installation scripts are provided with the xGDB distribution to expedite this process. Instances are generally populated with multiple feature components. Components are associated with each xGDB instance through an instance configuration file. Additional xGDB instances can be configured for additional species or separation of publicly accessible resources from proprietary systems. Each subsequent instance may share the initial xGDB core and any feature components installed therein. Instance based customization of feature component modules as described above may be used to distinguish further individual xGDB resources.Glycine max homeologous genomic sequences [Extensive options for customizing an xGDB instance are available. User interface properties such as color, image logos, and page layout are determined using a cascading style sheet. Modification of the default style sheet provided in the xGDB distribution allows an xGDB installer to quickly give any instance a unique look. Site navigation menus and controls can be customized using instance configuration files as well. These customization options are used with the xGDB instances at PlantGDB to provide additional informative content. This content includes species specific download pages; web pages relating relevant projects involving the use of xGDB, such as the characterization of U12-dependent introns using AtGDB; and links to relevant websites maintained by other research organizations. Third party groups and individuals are free to install, customize, and extend upon the xGDB system as provided for under the GNU general public license. In fact, one instance of xGDB has recently been applied to the annotation of equences .Arabidopsis and rice xGDB systems have been served from low-performance laptop computers to groups of 10 to 20 users with no noticeable performance loss, as well as from high-performance servers to the worldwide community. The xGDB systems interact with end-users through a combination of PHP and PERL generated web pages. Internet browsers that support HTML level 4, core JavaScript version 1.4 and higher, and Cascading Style Sheets level 2 and higher are required for complete user interface functionality. Default web pages have been design tested using Mozilla Firefox version 1.5.The xGDB distribution is available for download and requThe xGDB system provides an infrastructure for organization of genomic data, analysis of a wide range of inquiries about such data, and online publishing of both data and analysis results. The extensible design of xGDB provides a packaged solution to many types of research applications. In particular, xGDB is well suited for small to moderately sized research groups desiring local access to genomic data or an out-of-the-box system for analyzing emerging data.The xGDB system requires the following software packages: the Apache Web server , versionThe xGDB project is hosted on SourceForge.net, an online, open source development community. The complete xGDB distribution can be obtained from the xGDB project website . This si"} +{"text": "In addition, CMD displays data for three of the microsatellite projects that have been screened against a panel of core germplasm. The standardized panel consists of 12 diverse genotypes including genetic standards, mapping parents, BAC donors, subgenome representatives, unique breeding lines, exotic introgression sources, and contemporary Upland cottons with significant acreage. A suite of online microsatellite data mining tools are accessible at CMD. These include an SSR server which identifies microsatellites, primers, open reading frames, and GC-content of uploaded sequences; BLAST and FASTA servers providing sequence similarity searches against the existing cotton SSR sequences and primers, a CAP3 server to assemble EST sequences into longer transcripts prior to mining for SSRs, and CMap, a viewer for comparing cotton SSR maps.Gossypium spp.The collection of publicly available cotton SSR markers in a centralized, readily accessible and curated web-enabled database provides a more efficient utilization of microsatellite resources and will help accelerate basic and applied research in molecular breeding and genetic mapping in Comprehensive structural, functional and comparative studies of any genome are increasingly dependent upon the availability of an anchored physical map which shows the order of all genetic components in correspondence to their chromosomal localization. Anchoring of phenotypic information (such as trait or QTL) onto the physical map requires its integration with the genetic map of a genome which represents the relative positions of the genes and/or markers on chromosomes.The International Cotton Genome Initiative (ICGI) was launched to facilitate the development of a saturated and fully integrated genetic and physical map of cotton . A conseMicrosatellites consist of 1\u20136 repeating base pairs that are tandemly arranged in genomes . While tMicrosatellite markers are PCR-based, bi-parentally inherited, co-dominant markers. Polymerase chain reaction (PCR) products of different lengths can be amplified using unique primer pairs flanking the variable repeat microsatellite region after cloning and sequencing one allele. To develop microsatellite markers, primer sequences conserved between individuals and complementary to the microsatellite flanking sequences are identified by computer programs and synthesized.SSR loci tend to be both multiallelic and highly polymorphic for repeat number, which is easily scored and used for genotyping. SSRs are amenable to analysis on automated DNA sequencers, and can thus be adapted to high-throughput genotyping. SSRs are often markers of choice due to their abundance, co-dominance, reproducibility, and ease of use ,8. As miThe applications of microsatellites for plant breeders are numerous. They can be used for gene tagging and genome mapping, for selecting progeny before a desired phenotypic trait is expressed, for localizing qualitatively as well as quantitatively inherited traits, improving the efficacy of selective breeding (particularly for traits with low heritability or that can only be measured in one sex), genetic diversity studies, variety protection, gene and QTL analysis, pedigree analysis, and for introgressing novel genes into breeding germplasm from exotic germplasm .Gossypium germplasm to collect and integrate all the publicly available cotton microsatellite data in a centralized, curated, non-redundant online oracle database,(2) to provide access to the CMD standardized panel screened data,(3) to provide a set of comprehensive interface tools for rapid data retrieval,(4) to provide a suite of stand-alone microsatellite data mining tools,(5) to provide a communication portal for collaboration within the cotton research community.Currently, the database is composed of 14 tables which store all the data for the microsatellite projects including information on project collaborators, SSR-containing clones, sequences, primers flanking the SSRs, repeat motif, open reading frame position, genetic markers and maps, standardized panel varieties, and data homology, and publications. In a separate but linked database within CMD, the CMap schema consists of 16 tables including information about genetically mapped cotton SSRs. Data for cotton SSR markers and genetic maps, as well as panel screened cotton microsatellites, are submitted by researchers and then curated for any potential errors prior to uploading to the database using scripts written in Perl version 5.8.2. Web interfaces for database query and the query result pages are also developed in Perl.G. hirsutum small insert genomic library enriched for (GA/CT)n and (CA/GT)n inserts [In cotton, the first SSR markers were developed at the Brookhaven National Laboratory (prefix \"BNL\"). The 379 BNL microsatellites presented through CMD were derived from inserts . Later, inserts and 392 inserts microsat inserts . The 84 inserts , 1169 MU inserts and 1032 inserts ,21 micro inserts , mapping data, and any homology with known proteins. Marker data, primers, microsatellite sequences and standardized panel screened data are available for download directly from each project page as well as an overall downloads page. Currently, CMD contains information on 5,484 annotated cotton microsatellites which can be viewed and downloaded. Annotation of the sequences is periodically updated so that our data reflects changes in protein records in the NCBI GenBank non-redundant protein database.A microsatellite information page displays the sequence along with the repeat sequence and primers. The longest putative open reading frame (ORF) is also marked in color in the sequence along with the microsatellites. SSRs in the non-coding region tend to be more polymorphic and those in the coding region tend to be more transferable among species so the information of SSR position in a gene structure will be useful for marker development .Currently, 3,452 of the cotton microsatellites available through CMD have been checked for internal redundancy. Any of the following criteria were considered as redundant: 1) identical GenBank accession number; 2) completely identical primer pairs; 3) identical forward primers; 4) identical reverse primers; 5) forward primer identical to reverse and vice versa. From this analysis, 3,135 (90.8%) of the microsatellites checked were considered to be unique and were noted accordingly in the database.Gossypium genotypes for cotton microsatellite database (CMD) was established [Gossypium germplasm including cultivated and exotic cottons as shown in Table 1 (G. hirsutum) and AD2 (G. barbadense) species, respectively. Because TM-1 and 3\u201379 are also parents of a permanent RIL mapping population, they are essential for the integrated genome mapping and selection of the core reference markers. Acala Maxxa is California Upland cotton from which a BAC library is also constructed. DPL 458BR, Paymaster 1218BR, Fibermax 832, and Stoneville 4892BR are Upland cotton representatives with a significant acreage across the Cotton Belt and beyond. These Upland selections represent the contemporary Upland cotton variability and extend the Upland cotton diversity. They are often used as the National Variety Test (NVT) standards that provide a database of agronomic performance for any agronomic comparisons. Pima S-6 is the source of G. barbadense Pima germplasm breeding programs. G. arboreum (A2) and G. raimondii (D5) are representatives of A and D subgenomes, respectively. G. tomentosum (AD3) and G. mustelinum (AD4) are possible sources of introgression breeding programs \u00d7 VH8-4602 (G. barbadense)) \u00d7 Guazuncho2) [2: G. hirsutum race 'Palmeri\" \u00d7 G. barbadense Acc. \"K101\" [1: (TM-1(G. hirsutum) \u00d7 Hai7124 (G. barbadense)) \u00d7 TM-1) [G. hirsutum) \u00d7 3\u201379 (G. barbadense) [A genetically anchored physical map for cotton is being developed using cotton BAC libraries . Throughzuncho2) ,26; 2 - . \"K101\" ; 3 - BC1 \u00d7 TM-1) ,21; 4 - badense) . The ancThe CMD website is composed of general information pages Figure , includiThe initial SSR search result page displays SSR identifiers. The individual SSR entry links to a page where details of the SSR are displayed Figure with linCMD also provides a graphical tool CMap in which the cotton genetic maps Figure are dispThe CMD tools page provides access to an SSR server, a CAP3 Assembly server, and a sequence similarity server that includes BLAST and FASTA search tools.SSR analysis is performed using a modified version (SSR) of a Perl script SSRIT with parUsing the SSR server, users can upload a batch of sequences in FASTA format and select the motif type and repeat length to search. After job completion, users are redirected by email to a web page providing 1) a summary report of the SSR analysis, 2) a library file of the uploaded sequences, 3) a library file of the SSR containing sequences, and 4) an excel file of the individual properties of the SSR-containing clones. The individual properties include sequence name, length of the SSR-containing sequence, repeat(s) motif and number, SSR start/stop position, ORF start/stop position, primer pairs, SSR location relative to the ORF, and GC content of the sequence.To reduce the inherent redundancy and increase transcript length ESTs are routinely assembled into longer consensus sequences, also known as contigs. We have implemented the contig assembly program CAP3 as an onThe online BLAST and FASTA sequence similarity search servers allow users to perform homology searches between their sequences of interest and the annotated SSR sequences and primers in CMD. From the web interface, researchers can upload a file of sequences, select the search algorithm , the database and submit their job for processing. Once the job has completed an email is sent with a URL providing secure access to the results of the search. From the URL, users retrieve a summary of the search with the number of sequences that had matches with the database selected, an excel file containing the best match, any known function, match organism, match length, percent identity, expectation value, alignment length, and start and stop alignment positions. Our sequence similarity server, specifically designed for CMD researchers, will help users compare new sequences and primers against existing microsatellites and help decrease redundancy of effort in developing new markers. As we migrate the sequence similarity servers to a computational cluster, we plan to add the following databases: NCBI cotton ESTs, TIGR cotton gene indices, NCBI cotton genomic sequences and NCBI cotton protein sequences.Future development will focus on the establishment of a standard nomenclature of cotton SSRs, adding new microsatellite data, improving the tools and functionality of the web interface, such as an advanced search site with options for search/display categories, full sequence processing facilities for cotton researchers, and a quarterly newsletter for the cotton community. The annotation of the SSRs with known homology will include further classification using the gene ontology terms associated with the matching sequences in the Swissprot database. When the physical map is available, users also will be able to retrieve the anchored BAC clones containing the SSRs of interest through the anchored BACs page in the map viewer. Data that are currently scheduled to be added in the near future include 800 BAC-derived cotton genomic SSRs and 500 cotton EST-SSRs from the public domain, and 200 SSRs from private companies.The CMD has been initiated to provide researchers, engaged worldwide in cotton research, with centralized access to microsatellite markers, an invaluable resource for basic and applied research in cotton breeding. As such, the CMD serves the cotton community as a major repository of the publicly available cotton microsatellite data and a unique repository for the CMD standardized panel screened data, a key tool for systematic characterization of the SSR markers developed for cotton. Access to this data is provided through integrated web tools which allow users to directly access individual or combined project data via search interfaces which provide download and visualization of microsatellites, their flanking primers, open reading frames (ORFs), and SSR genetic maps. CMD also provides a suite of online tools for data analysis of new and existing microsatellites through its SSR, CAP3, and FASTA/BLAST servers. Overall, the CMD serves as a major resource for the international cotton community, and can be viewed as an important vehicle toward increased collaboration among cotton scientists.. The CMD is a relational database implemented using the Oracle Relational Database Management System version 9.2.0. Users can subscribe to the CMD mailing list, but registration is not required to use the CMD.CMD is publicly available at the URL BAC \u2013 Bacterial Artificial Chromosomest generationBC1 \u2013 Backcross 1BLAST \u2013 Basic Local Alignment Search ToolCAP3 \u2013 Contig Assembly ProgramCIRAD \u2013 Centre International en Recherche Agronomique pour le D\u00e9veloppementCIR \u2013 CIRadCM \u2013 Cotton MicrosatellitesCMap \u2013 Comparative MapEST \u2013 Expressed Sequence TagEXP \u2013 Expectation ValueFASTA \u2013 Fast All alignment search toolFLIP \u2013 FLexible In-system ProgrammerJESPR \u2013 Jenkins, El-Zik, Saha, Pepper, Reddy microsatellite repeatsGossypium hirsutum EST-SSRMGHES \u2013 Mississippi MUCS \u2013 Microsatellite Ulloa Complex Sequence repeatsMUSB \u2013 Microsatellite Ulloa Simple BAC repeatsMUSS \u2013 Microsatellite Ulloa Simple Sequence repeatsNAU \u2013 Nanjing Agricultural UniversityQTL \u2013 Quantitative Trait LociRIL \u2013 Recombinant Inbred LineTMB \u2013 TM-1 genetic standard BAC/BIBAC libraries microsatellite repeatsAB participated in the database and interface design, performed general CMD data collection, organization and curation. CJ, SM, PY participated in the database and interface design and construction, and developed scripts for database upload and sequence processing. SJ implemented CMap, uploaded mapping data and was involved with the database schema design and implementation, SF managed the oracle system and was involved in database schema design and tool development, MS performed all homology searches and was involved with database design and construction, RE helped design and implement the SSR, FASTA, BLAST and CAP3 servers, JS and BS screened the cotton SSRs against the CMD standardized panel. MP participated in the development of the MUSB microsatellite project and participated in the database and interface design. JML developed and provided data for the CIR project and participated in the CMD general data collection and organization. JY established, maintained, and distributed the 12-genotype CMD panel DNA stocks as well as developed and provided data for the TMB project and participated in the general coordination of the project. MU developed and provided data for the MUSS/MUCS and MUSB projects, participated in collection and organization of mapped SSR data presented in the CMD. SS was the lead scientist in developing MGHES and provided data for the MGHES and JESPR projects and was involved in the general coordination of the project. BB and SL developed and provided data for the BNL project, critically reviewed and contributed to database and manuscript development. TZ developed and provided data for the NAU project, critically reviewed and contributed to database and manuscript development. AP participated in the development of the JESPR project, critically reviewed and contributed to database and manuscript development. DF performed redundancy analysis of the CMD microsatellites and was involved in the general coordination of the project. JT participated in the development of the MUSB microsatellite project. SK and JJ served on the CMD Advisory Board, critically reviewed and contributed to database and manuscript development. RC conceived and performed general coordination of the project. DM supervised the project and was involved with all aspects of database design, construction and implementation. All authors read and approved the final manuscript."} +{"text": "Much work in immunoinformatics has involved the algorithmic prediction of peptide binding affinity to various MHC-I alleles. A number of tools for MHC-I binding prediction have been developed, many of which are available on the web.Peptides derived from endogenous antigens can bind to MHC class I molecules. Those which bind with high affinity can invoke a CD8We hypothesize that peptides predicted by a number of tools are more likely to bind than those predicted by just one tool, and that the likelihood of a particular peptide being a binder is related to the number of tools that predict it, as well as the accuracy of those tools. To this end, we have built and tested a heuristic-based method of making MHC-binding predictions by combining the results from multiple tools. The predictive performance of each individual tool is first ascertained. These performance data are used to derive weights such that the predictions of tools with better accuracy are given greater credence. The combined tool was evaluated using ten-fold cross-validation and was found to signicantly outperform the individual tools when a high specificity threshold is used. It performs comparably well to the best-performing individual tools at lower specificity thresholds. Finally, it also outperforms the combination of the tools resulting from linear discriminant analysis.A heuristic-based method of combining the results of the individual tools better facilitates the scanning of large proteomes for potential epitopes, yielding more actual high-affinity binders while reporting very few false positives. The major histocompatibility complex (MHC) is a set of genes whose products play a crucial role in immune response. Peptides derived from the proteasomal degradation of intracellular proteins are presented by MHC class I molecules to cytotoxic T lymphocytes (CTL) -3, and rOnly 0.5% of peptides are estimated to bind to a given MHC-I molecule, making this the most selective step in the recognition of intracellular antigens ,5. GivenMany tools for performing such predictions, of varying quality, are available. We hypothesize that greater predictive accuracy can be achieved by combining the predictions from several of these tools rather than using just one tool. Further, contributions from individual tools should be related to their accuracy. To test this hypothesis, we have built a prediction tool which assigns a \"combined score\" to each peptide in a given protein by taking into account the predictive performance of each tool, and the score given by that same tool to a given peptide. We also compare our technique with combined predictions made using linear discriminant analysis, a standard statistical method for combining variables to distinguish two groups . In this paper, the acronym \"HBM\" will refer to our heuristic-based method and \"LDA\" will refer to the predictor built using linear discriminant analysis.AROC value for each tool is also given; these values are very similar, but not completely identical, to those given by the authors of the community binding resource; the small discrepancies are likely due to the use of differing methods of calculating the area under the ROC curve. Individual tool performance data for the HLA-B*3501 and H-2Kd peptides from the community binding database, as well as for the HLA-A*0201 peptides gathered from the literature, are shown in Tables Table The HBM and LDA were evaluated using ten-fold cross-validation on the same four datasets as the individual tools.The HBM requires that an individual tool specificity parameter be chosen such that the tools' sensitivities at that specificity can be used as the weights in equation 1. The performance of the HBM was determined using individual tool specificities of 0.99, 0.95, 0.90, and 0.80. In general, it was found that using 0.99 individual tool specificity resulted in the best performance, while the use of lower individual tool specificity parameters resulted in somewhat weaker performance. Thus, all of the HBM performance data described below were obtained using 0.99 individual tool specificity. Table For two of the three alleles, the HBM showed marked improvements in sensitivity at high specificity compared with the best-performing individual tools. The sensitivity of the HBM at 0.99 specificity for HLA-A*0201 was 0.40, a large increase over NetMHC ANN, whose sensitivity of 0.29 was the best among the individual tools. For HLA-B*3501, the HBM sensitivity was 0.31 at a specificity of 0.99, while the highest sensitivity obtained by an individual tool was 0.24. The HBM showed similarly strong performance when tested using the literature-derived HLA-A*0201 data, achieving a sensitivity of 0.27, compared with 0.19 for the best-performing individual tool. For H-2Kd, however, the HBM was outperformed at 0.99 specificity by the ARB matrix tool, which had a sensitivity of 0.50 versus 0.47 for the HBM. We note, however, that ARB Matrix was trained using binders from the community binding database, so its performance on the community datasets is likely inflated -1.p (between 0 and 1) to log(p/(1 - p)). For a variable y which is restricted between a and b, a \"scaled logit\" can be calculated via log((y - a + \u03b5)/(b - y - \u03b4)), where \u03b5 and \u03b4 are small adjustments to avoid zeros. \u03b5 = (y- - a)/2 and \u03b4 = (b - y+)/2, y- and y+ being the smallest and largest observed values greater or less than a or b, respectively. The actual performance of the linear discriminant on the transformed scores was estimated using ten-fold cross-validation. Computations were done using S-PLUS version 7.0.0. Figures were created with MATLAB 7.A number of the tools displayed notably non-normal distributions. Most of these were highly skewed, but became close to normal when transformed to logarithms. The scores of three tools had sigmoidal distributions. These became approximately normal when converted to scaled logits. A \"logit\" is a transformation of a probability Except for the H-2Kd dataset, the cross-validated specificities fell short of the nominal ones. To realize specificities of 0.99 and 0.90, the threshold was adjusted to a nominal specificity such that the cross-validated values were as close as possible to the target values. Figure LDA \u2013 linear discriminant analysisHBM \u2013 heuristic-based methodThe authors have no conflict-of-interest with respect to this work. In particular, they have no direct connection with any of the researchers involved with the binding prediction tools studied.BT performed the design, programming work, and evaluation of the HBM. MB performed the linear discriminate analysis work. AK proposed the original idea, provided bioinformatics expertise, contributed to the methodology, and supervised the work. All three authors contributed to the paper, with a majority written by BT.Literature-derived HLA-A*0201 binders and non-binders. List of HLA-A*0201 binding and non-binding peptides gathered from the literature. The papers from which these peptides were derived are cited in the text.Click here for fileHLA-B*3501 LDA Q-Q plot. Q-Q plot showing distribution of LDA scores for the H-2Kd dataset from the community binding resource. The horizontal axis has been scaled according to normal probabilities, so that points from a normally distributed variable would fall along a straight line (shown in blue). Scores lying above the thresholds indicated would be classified as epitopes. The realized sensitivity of 0.44 for a specificity of 0.95 is indicated as the proportion of epitopes whose scores lie above the threshold of 0.95. Of the four datasets used, this one deviates most strongly from normality.Click here for fileH-2Kd LDA Q-Q plot. Q-Q plot showing distribution of LDA scores for the H-2Kd dataset from the community binding resource. The horizontal axis has been scaled according to normal probabilities, so that points from a normally distributed variable would fall along a straight line (shown in blue). Scores lying above the thresholds indicated would be classified as epitopes. The realized sensitivity of 0.42 for a specificity of 0.99 is indicated as the proportion of epitopes whose scores lie above the threshold of 0.99. Only the nominal values for specificity are used, since the actual ones coincide or are better.Click here for fileHLA-A*0201 (literature) LDA Q-Q plot. Q-Q plot showing distribution of LDA scores for the HLA-A*0201 dataset derived from literature. The horizontal axis has been scaled according to normal probabilities, so that points from a normally distributed variable would fall along a straight line (shown in blue). Scores lying above the thresholds indicated would be classified as epitopes. The realized sensitivity of 0.33 for a specificity of 0.95 is indicated as the proportion of epitopes whose scores lie above the threshold of 0.95. Of the four datasets used, this one best fits the normality assumption.Click here for file"} +{"text": "The procedure was based on the flow-batch approachimplemented employing multicommutation. The photometric detection was carried outusing a homemade LED-based photometer. The mixing device, LED, and photodetectorwere attached to the titration chamber in order to form a compact and small-sized unit. The flow system comprised an automatic injector and three-way solenoid valves, which werecontrolled by a microcomputer through an electronic interface card. The software, writtenin Quick BASIC 4.5, was designed with abilities to accomplish all steps of the titrationprocedure including data acquisition and real-time processing to decide about the course of titration in the following step and so forth, until the titration endpoint was reached. The usefulness of the proposed titration system was demonstrated by analyzing red wine samples. When results were compared with those obtained using the AOAC referencemethod, no significant difference was observed at the"} +{"text": "Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA. The homotrimeric PNPase engages RNase E on the periphery of its ring-like architecture through a pseudo-continuous anti-parallel \u03b2-sheet. A similar interaction pattern occurs in the structurally homologous human exosome between the Rrp45 and Rrp46 subunits. At the centre of the PNPase ring is a tapered channel with an adjustable aperture where RNA bases stack on phenylalanine side chains and trigger structural changes that propagate to the active sites. Manganese can substitute for magnesium as an essential co-factor for PNPase catalysis, and our crystal structure of the enzyme in complex with manganese suggests how the metal is positioned to stabilise the transition state. We discuss the implications of these structural observations for the catalytic mechanism of PNPase, its processive mode of action, and its assembly into the RNA degradosome.Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Messenger RNA decay is the counterweight of transcription in the dynamic expression of genetic information. The regulated turnover of transcripts contributes to homeostasis and to timely and efficacious response to environmental change.,Salmonella sp., PNPase regulates the expression of small non-coding RNAs that control expression of outer-membrane proteins.Salmonella enterica,Yersinia.Escherichia coli, PNPase is involved in the quality control of ribosomal RNA precursors,PNPase is found in diverse species, with homologues identified in eubacteria and eukaryotic organelles such as the chloroplast and mitochondria, although curiously there is no known archaeal PNPase.Streptomyces antibioticus reveals a homotrimeric subunit organisation that encloses a central channel proposed to form the pathway for RNA substrates to enter the active sites.S. antibioticus PNPase structure suggests that only the C-terminal RNase PH domain has the capacity to catalyse phosphorolysis.,,The crystal structure of PNPase from the Gram-positive bacterium S. antibioticus PNPase and, like the KH domain, the S1 domain is found in many proteins that interact with single-stranded nucleic acids.E. coli PNPase lacking both those RNA-binding domains. This truncated PNPase, which we refer to here as the PNPase core, was found to have reduced RNA-binding affinity while retaining phosphorolytic activity.Sequence analysis suggests that the duplication of the RNase PH-like domain and the organisation of other sub-domains of s PNPase b is consE. coli, a small proportion of PNPase is associated with the multi-enzyme RNA degradosome . S. antibioticus PNPase has four inter-strand loops that are absent or truncated in the E. coli enzyme. Helix H4 of S. antibioticus PNPase is absent from the E. coli enzyme under four different conditions, providing several independent views of the micro-domain\u2013enzyme interaction . The RNahe metal . The locBecause two RNase PH-like domains of the PNPase protomer are related by an internal pseudo-dyad, there are potentially two sites within each protomer that could form an extended sheet-like interaction with the RNase E micro-domain; however, only the amino-terminal RNase PH sub-domain forms an interaction with RNase E. The observed stoichiometry of one PNPase monomer binding to one RNase E micro-domain in the crystal structures is consistent with data from mass spectrometry,An earlier study found that PNPase core and full-length PNPase bind to RNase E with roughly equal affinity.E. coli RNase E micro-domain-PNPase interaction and the human Rrp45-Rrp46 interaction within the exosome is likely to represent convergent evolution and highlights how the exposed peptide backbone of a \u03b2-sheet may be a favoured site for protein\u2013protein interactions.Remarkably, the interaction between the RNase E micro-domain and PNPase closely resembles the contact made between the RNase PH-like subunits Rrp45 and Rrp46 in the human exosome b. A portIn our screen for well diffracting co-crystals of PNPase core with RNA, several modified RNA oligonucleotides were tested, and well diffracting co-crystals were obtain for a 12-mer RNA in which the sequence originates for a preferred cleavage site for RNase E . The RNAS. antibioticus and E. coli PNPases identified two constricted points in the channel.,E. coli residues F77-F78-R79-R80 the active site.E. coli enzyme .The FFRR channel-loops in the RNA-free ed state b. In thiThe functional importance of the central aperture residues were verified earlier. Substitution of R79 and R80 in the FFRR loop had little apparent effect on activity but caused the full-length PNPase to stall on RNA oligomers shorter than eight nucleotides.S. antibioticus PNPase and archaeal and human exosomes at the level of protomer fold and quaternary structure.,E. coli PNPase core, the archaeal exosomes engage RNA at the constriction in the central channel.S. solfataricus archaeal exosome and E. coli PNPase core confirms the expected structural homology. While RNA binds to the central aperture in both bacterial PNPase and archaeal exosome, it is seen at different depths in the channel , with the key interaction occurring near an incomplete helix , and since Mn2+can be more readily identified in difference maps and anomalous Fourier syntheses, we prepared RNA-free co-crystals of the PNPase core in the presence of 20\u00a0mM manganese acetate that forms an aperture at the mouth of the central channel near the S1/KH RNA-binding domains. Mutation of R79 and R80 from this loop affect RNA binding affinity and catalytic activity of PNPase,E. coli PNPase and PNPase core in the RNA-free forms shows that the central aperture is expanded in the latter.E. coli and S. antibioticus PNPase, it is difficult to envisage how these domains might communicate with the core to affect the aperture size. However, it is possible that RNA binding by the KH and S1 domains may itself contribute structural changes at the aperture.Our structure of the PNPase core in complex with modified RNA shows that F77 of each PNPase core monomer makes an aromatic stacking contact with an RNA base. F77 is part of the conserved FFRR loop has little apparent effect on activity, it causes the full-length PNPase to stall on RNA oligomers shorter than eight nucleotides.E. coli PNPase core and Mn2+reveals that the metal is coordinated by residues D486, D492 and K494. These residues are also conserved in human PNPase .P. abyssi exosome shows that the scissile phosphate of RNA is spatially coincident with one of two orientations for the \u03b1-phosphate of ADP, whereas the \u03b2-phosphate is positioned at a binding site for inorganic phosphate. Strikingly, an overlay of ADP and RNA forms resembles the pentavalent phosphate transition state proposed for many phosphotransfer reactions. Guided by this overlay, a model for the transition state of an RNA substrate under attack by PNPase was prepared. Firstly, the phosphate for the transition state was placed at the mean position of the phosphate atoms in the ADP and RNA-bound forms of the P. abyssi exosome. Secondly, the oxygen atoms that formed a trigonal planar arrangement were selected from either structure to generate a chimeric structure that resembles the transition state. Lastly, we docked the chimeric RNA structure from the P. abyssi exosome structures onto the corresponding position in our PNPase core-manganese structure. The overlay shows that the metal is in a good position to support the proposed transition state . Our crystallographic data presented here now reveal how one of these micro-domains from RNase E recognises PNPase core. A segment of 20 amino acids from the RNase E C-terminal domain forms hydrogen-bonding interactions with the exposed ridge of the amino-terminal RNase PH domain of PNPase in an extended \u03b2-sheet and also makes van der Waals interactions through a small helical segment. The interaction is at a distance from the active site, and it does not affect enzyme activity .Earlier biophysical and computational analyses have indicated that the RNA degradosome is built upon interactions of its canonical components with small peptide micro-domains lying within the C-terminal domain of RNase E.Streptomyces coelicolor has been reported to form a stable complex with PNPaseE. coli RNase E 1021-1061, we suggest that the interaction of S. coelicolor RNase E and PNPase could be mediated by a sheet\u2013strand interaction similar to that used by the E. coli proteins.Remarkably, a similar interaction occurs between the RNase PH-like subunits Rrp45 and Rrp46 in the human exosome, where the C-terminal tail of Rrp46 plays the analogous role of RNase E micro-domain to form a pseudo-continuous \u03b2-sheet with the Rrp45 subunit. This similarity is likely to represent convergent evolution and emphasises how the exposed surface of a sheet may be a highly favoured \u201chot-spot\u201d site for protein\u2013protein interactions. Indeed, molecular recognition through peptide-sheet interaction is seen in several other protein\u2013peptide complexes.in vivo the degradosomes are not self-contained and are linked together in a more complex incommensurate oligomer. In such a case, the micro-domain of one degradosome is envisaged to interact with an oligomeric partner in a neighbouring degradosome. The PNPase composition of degradosomes isolated from cells can change, depending on physiological conditions,Using calorimetry, we observe a 1:1 binding stoichiometry of PNPase core to the RNase E micro-domain; moreover, the observed binding affinity is roughly 0.9\u00a0\u03bcM, which is weak for a macromolecular interaction. The interaction is likely to be stronger in the context of full-length RNase E, which is a tetramer, so that the PNPase binding sites will be concentrated locally. In this regard, it is noteworthy that the observed ratio of 1:1 PNPase:RNase E represents a mismatch of the molecular symmetries of the trimeric PNPase and the tetrameric RNase E. One potential resolution of the mismatch is for the formation of a greater complex of 12 subunits of both proteins, comprising three RNase E tetramers and four PNPase trimers.E. coli PNPase \u0394K\u0394H1 (the PNPase core) was kindly provided by George Mackie (University of British Columbia). The plasmid was transformed into E. coli BL21 (DE3), and the core protein expressed by auto-induction.The expression vector encoding \u20131061 was synthesised by Clonestar Biotech Ltd, Czech Republic, desalted using a PD10 column , lyophilised, re-suspended in 20\u00a0mM Tris\u2013HCl pH 7.5, 50\u00a0mM NaCl and stored at \u201380\u00b0C. The RNA oligomer 5\u2032-GGGACAGUAUUG-3\u2032 with 5\u2032-monophosphate and O2\u2032-methyl groups was synthesised by Charles Hill (PNAC facility), de-salted, HPLC-purified using an ion-exchange column , desalted by preparative G25 chromatography, and lyophilised for storage.RNase E micro-domain corresponding to residues 10212+co-crystals were prepared using a reservoir buffer composed of 2.5\u00a0M NaCl, 9 % (w/v) PEG 6000, 20\u00a0mM sodium citrate and 20\u00a0mM manganese acetate tetrahydrate. The crystals were transferred briefly into reservoir solution supplemented with 20\u201325 % glycerol as cryoprotectant before flash-freezing in liquid nitrogen.PNPase complexes with RNase E were prepared by mixing 165\u00a0\u03bcM PNPase core with 246\u00a0\u03bcM RNase E micro-domain (1021-1061) and kept on ice for 30\u00a0min. To prepare the RNA complexes, 330\u00a0\u03bcM RNA was added to the PNPase/RNase E micro-domain complex and incubated for a further 15\u00a0min. Crystals were grown at 20\u00a0\u00b0C by the hanging-droplet, vapour-diffusion method by mixing 1\u00a0\u03bcl of complex with 1\u00a0\u03bcl of reservoir solution. Crystals for the PNPase core/RNase E micro-domain crystals were grown using a reservoir solution containing 0.2\u00a0M ammonium nitrate, 20 % (w/v) PEG 3350. Crystals for the PNPase core/RNase E micro-domain-RNA complex were produced from a reservoir solution containing 0.2\u00a0M di-ammonium hydrogen citrate, 17% PEG 3350. The measured pH was \u223c4.5. The optimal reservoir buffer for the PNPase core/RNase E micro-domain-RNA-tungstate crystals was composed of 0.2\u00a0M di-ammonium hydrogen citrate, 17 % PEG 3350, pH \u223c4.5, 50\u00a0mM disodium tungstate. Crystals for the PNPase core/RNase E micro-domain-MnS. antibioticus PNPase structure, PDB entry 1E3H.All X-ray diffraction data were collected at 100 K from cryoprotected crystals. X-ray diffraction data for the PNPase/D-micro-domain complex, the PNPase/D-micro-domain\u2013RNA complex and the PNPase\u2013tungstate\u2013RNA complex were collected at the microfocus station ID23-2 at the ESRF facility in Grenoble, France. Data for the manganese crystals were collected with a Raxis IV and Cu rotating anode source. The data were processed and scaled using the HKL package,E. coli PNPase-RNase E micro-domain-RNA complex was used as the search model of the PNPase core/Mn2+. The helical domain for this structure . PNPase core and RNase E micro-domain (1021-1061) were dialysed extensively against 10\u00a0mM Tris pH 7.5, 150\u00a0mM NaCl, 1\u00a0mM MgCl3GCM for the PNPase/RNase E micro-domain/RNA tetragonal crystal form (RCSB ID 051694). The accession number of the PNPase core with RNase E peptide is code 3GLL (RCSB 052013), for the complex with manganese is code 3GME (RCSB 052041), and for the complex with RNA and tungstate is 3H1C (RCSB 052567).Coordinates and structure factors have been deposited in the Protein Data Bank with accession number"} +{"text": "Burkholderia pseudomallei, is a common cause of sepsis in northeast Thailand. In white North Americans, common functional genetic variation in TLR1 is associated with organ failure and death from sepsis. We hypothesized that TLR1 variants would be associated with outcomes in Thais with melioidosis. We collated the global frequencies of three TLR1 variants that are common in white North American populations: rs5743551 (-7202A/G), rs4833095 (742A/G), and rs5743618 (1804G/T). We noted a reversal of the minor allele from white North American subjects to Asian populations that was particularly pronounced for rs5743618. In the Utah residents of European ancestry, the frequency of the rs5743618 T allele was 17% whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic association study in 427 patients with melioidosis to determine the association of TLR1 variation with organ failure or death. We genotyped rs5743551 and rs4833095. The variants were in high linkage disequilibrium but neither variant was associated with organ failure or in-hospital death. In 300 healthy Thai individuals we further tested the association of TLR1 variation with ex vivo blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly associated with blood cytokine responses induced by Pam3CSK4. We identified additional common variation in TLR1 by searching public databases and the published literature and screened three additional TLR1 variants for associations with Pam3CSK4-induced responses but found none. We conclude that the genetic architecture of TLR1 variation differs substantially in southeast Asians compared to other populations and common variation in TLR1 in Thais is not associated with outcome from melioidosis or with altered blood responses to Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory host response and determinants of sepsis in southeast Asian populations.Melioidosis, infection caused by the Gram-negative bacterium Burkholderia pseudomallei - is the second most common cause of bacteremia and a frequent cause of sepsis The global burden of sepsis is estimated at up to 19 million cases per year TLR4 variants are associated with infection TLR5 is highly associated with survival in individuals with melioidosis Genetic variation accounts for susceptibility to and outcome from infectious disease, and provides a window into mechanisms that underlie the complex pathophysiology of sepsis TLR1 variants have been described as associated with sepsis, although the relationship is complex. rs5743551 (-7202A/G) is upstream of TLR1. In white North American subjects, this polymorphism tags two non-synonymous polymorphisms: rs5743618 and rs4833095 TLR1, changes a serine to an isoleucine at amino acid 602. The T allele has been demonstrated by multiple authors to be relatively hypermorphic TLR1 variants is markedly different around the world TLR1 is another sensor in the TLR family that forms a heterodimer with TLR2 and facilitates innate immune activation upon ligation of bacterial cell wall components such as lipopeptides, peptidoglycan, and lipotechoic acid B. pseudomallei is recognized by TLR2/1, inducing rapid upregulation of the innate immune response TLR1 may modulate host defense in melioidosis. In light of the data showing an important role for TLR1 in human sepsis and as a trigger for B. pseudomallei-induced immune system activation, we undertook an analysis of the association of common hypermorphic TLR1 genetic variants with outcome in a cohort of Thai subjects with culture-proven melioidosis. We also analyzed the association of TLR1 variants with blood cytokine responses to a TLR1 agonist in healthy Thai subjects. We hypothesized that hypermorphic TLR1 variation would be associated with altered outcome, including death and organ failure. Notably, we found that the genetic architecture of functional TLR1 variation is substantially different in southeast Asian populations, that common TLR1 variation in Thais is not hypermorphic, and that common variation in TLR1 in Thais is not associated with outcome in melioidosis. Our data suggest that immunogenetic determinants of outcome from Gram-negative infection in Thais differ from previously described determinants in white North American subjects with sepsis.B. pseudomalleiB. pseudomallei from a sample collected by the study team or independently by hospital clinicians. Demographic and clinical data from enrolment until discharge from hospital was recorded by the study team. All patients included in this analysis were Thai. Outcomes for this study were organ failure or death. Organ failure was defined as respiratory failure or shock. The definition for respiratory failure was hypoxia (PaO2 <60 mmHg) or hypercapnia (PaCO2 >50 mmHg) in conjunction with acidaemia (blood pH <7.30) or requirement for mechanical ventilation. The definition of shock was hypotension despite adequate fluid resuscitation or need for vasopressors to maintain systolic blood pressure >\u200a=\u200a90 mmHg or mean arterial pressure >\u200a=\u200a70 mmHg. Death was defined as in-hospital death or discharge in a moribund condition for palliative care at home. Written informed consent for enrollment into clinical studies of melioidosis was obtained from subjects or their representatives at the time of recruitment.Subjects with melioidosis were identified among inpatients at Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand from 1999 to 2005. A study team screening patients cultured blood, urine, and other relevant samples (e.g. abscess aspirates) for Three-hundred healthy subjects donating blood at the blood donation center at Sappasithiprasong Hospital in 2010 were recruited for participation as previously described The Ethical Review Committee for Research in Human Subjects, Ministry of Public Health, Thailand, and the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, and the University of Washington Human Subjects Division Institutional Review Board approved the consent procedure and protocol of these studies.2 for 6 h before being spun down and plasma removed and frozen at \u221280\u00b0C. IL-6, IL-8, TNF-\u03b1, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1\u03b2 were later assayed in duplicate on a multiplex bead system using reagents from R&D Systems . A complete blood count with differential was performed in the hospital clinical laboratory for each subject at the time of phlebotomy.Three hundred and eighty microliters of fresh whole blood in citrate mixed 1\u22361 with RPMI media was added to pre-prepared plates containing 20 \u00b5L of Pam3CSK4 for a final concentration of 100 ng/mL. Pam3CSK4 is a specific agonist for TLR2/1. Plates were incubated at 37\u00b0C on a shaking incubator under 5% COTLR1 polymorphisms rs5743551 (-7202A/G) and rs4833095 (742A/G) to genotype in the melioidosis cohort using an allele-specific primer extension method with reads by a MALDI-TOF mass spectrometer TLR1 variants were identified as follows: SNP selection of non-synonymous TLR1 coding variants was performed by searching the HapMap project database (http://hapmap.ncbi.nlm.nih.gov) and based on functional prediction using a FastSNP analysis (http://fastsnp.ibms.sinica.edu.tw) TLR1 SNPs were selected from the Han Chinese in Beijing and Japanese in Tokyo populations in the HapMap database for variants with a minor allele frequency (MAF) at least 2% using the HapMap tag-SNP picker option. Genotyping was performed using Fluidigm SNPtype assays on the Biomark real time PCR system.DNA was extracted from whole blood of patients with melioidosis using Nucleon BACC3 kits or from whole blood of healthy donors using the QIAamp DNA Blood Midi Kit . We selected TLR5 variant rs5744168 (1174C/T) that we have previously shown to be associated with outcome from melioidosis TLR1 genotype associations. Survival analyses were performed with the log rank test. Healthy subject plasma cytokine levels were normalized to monocyte count and log10 transformed before analysis by linear regression, adjusting for age, gender, and batch. Analyses were performed with Stata version 11.2 . P values \u22640.05 were considered significant.Deviation from Hardy-Weinberg equilibrium was calculated for each variant using the exact test. In the melioidosis cohort, the power to detect an association between genotype and death was estimated to be 99% for a risk allele frequency of 0.45-0.55, assuming a recessive model and genotype relative risk of 2, 40% mortality in the population, 100 nonsurvivors, 300 survivors, and alpha \u200a=\u200a0.05 TLR1 variants in the published literature or publically accessible databases . Following the genetic models described by Wurfel and Thompson Given the different distribution of 2\u200a=\u200a0.91). Genotype and allele frequencies for these variants are given in As rs5743551 is associated with altered immune responses by whole blood in white American and African American healthy subjects TLR1 variants in Asian subjects that might underlie the variable innate immune response of Thai subjects to Pam3CSK4. We identified TLR1 region variants as described in the methods and genotyped seven additional polymorphisms . Genotype and allele frequencies are given in 2\u22650.96) so we se 2\u22650.96) .TLR1 in Thais is not associated with altered inflammatory responses to Pam3CSK4 in blood or with outcome from melioidosis, a common cause of sepsis in northeast Thailand. Our findings also confirm that the genetic architecture of functional TLR1 variation is substantially different in southeast Asians compared to other global populations.The results of this investigation show that common variation in TLR1 variants with clinical outcome from one of the major Gram-negative causes of sepsis in northeast Thailand. Immunoassays in subjects recruited from the same population can provide corroborating functional evidence of inflammatory effects attributable to genotype. However, our data do not implicate known common genetic variants in TLR1 in modulating the host response clinically or under experimental conditions.Sepsis remains a vexing syndrome to identify and treat TLR1 genetic architecture across populations.In white Americans, rs5743551 is in moderate to high LD with the non-synonymous coding variants rs5743618 and rs4833095. All three variants have been associated with greater inflammation or death from sepsis TLR1 variation tagged by rs5743551. rs4833095 is a possible candidate that is in fact associated with mortality in this Gram-positive trauma-related sepsis population TLR5 polymorphism, rs5744168, is strongly protective against death in this cohort, but is not associated with susceptibility to melioidosis Despite the documented functional effects of rs5743618, there is a stronger association of the tag SNP rs5743551 G allele with death from sepsis than for the rs5743618 T allele in white North Americans In addition to the absence of an association of rs4833095 with death from melioidosis in Thais, our present functional studies do not support an effect of rs4833095 on the host inflammatory response. Notably, the complete lack of effect on Pam3CSK4-induced cytokine release by rs4833095 in Thai subjects stands in contrast to recent work by Mikacenic et al TLR1 variation did not reveal any other variants associated with altered inflammatory response to Pam3CSK4. Our approach was restricted to previously described variation, however, and it is clear that there is marked variation in the genetic architecture of TLR1 across populations, possibly driven by infectious disease TLR1 genetic variation in Asians remains to be discovered, and that this may regulate outcomes in sepsis. Although current genome wide association studies chips are characterized by a relative paucity of Asian variants, ongoing comprehensive sequencing efforts across a variety of populations are facilitating identification of potentially clinically relevant variants in relatively understudied populations Our screening analysis of additional common TLR1 polymorphisms in sepsis noted a relationship between frequency of Gram-positive infection and rs5743551 genotype B. pseudomallei in vitro While melioidosis is representative of Gram-negative sepsis, a prior study of Potential limitations to all genetic association studies include population stratification due to ethnic admixture and other unmeasured confounding. Previously, however, we have not observed substantial population stratification in our Sappasithiprasong cohort TLR1 genetic variation with outcomes from sepsis. We are also unable to identify functional effects of selected common TLR1 variants in Thais. We confirm substantial differences in the genetic architecture of TLR1 variation of Thais compared to other populations. These findings underscore the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory host response and determinants of sepsis in southeast Asian populations.In conclusion, in our cohort of Thai subjects with melioidosis we do not confirm the previously described associations in Spanish and white North American populations of Table S1TLR1 variant genotype frequencies in selected global populations.(DOCX)Click here for additional data file.Table S2Minor allele frequencies and association of TLR1 variants with cytokine induced by stimulation of whole blood with Pam3CSK4 in healthy Thai subjects.(DOCX)Click here for additional data file."} +{"text": "Patients in the intensive care unit (ICU) are at risk of developing of intra abdominal hypertension (IAH) and abdominal compartment syndrome (ACS).Aim: This review seeks to define IAH and ACS, identify the aetiology and presentation of IAH and ACS, identify IAP measurement techniques, identify current management and discuss the implications of IAH and ACS for nursing practice. A search of the electronic databases was supervised by a health librarian. The electronic data bases Cumulative Index of Nursing and Allied Health Literature (CINAHL); Medline, EMBASE, and the World Wide Web was undertaken from 1996- January 2011 using MeSH and key words which included but not limited to: abdominal compartment syndrome, intra -abdominal hypertension, intra-abdominal pressure in adult populations met the search criteria and were reviewed by three authors using a critical appraisal tool. Data derived from the retrieved material are discussed under the following themes: (1) etiology of intra-abdominal hypertension; (2) strategies for measuring intra-abdominal pressure (3) the manifestation of abdominal compartment syndrome; and (4) the importance of nursing assessment, observation and interventions. Intra-abdominal pressure (IAP) and abdominal compartment syndrome (ACS) have the potential to alter organ perfusion and compromise organ function. The importance of the diagnosis and management of intra-abdominal hypertension (IAH) and abdominal compartment syndrome (ACS) is increasingly recognised. These conditions can alter organ perfusion and as a consequence end organ function. Complications resulting from IAH and ACS can be life threatening to critically ill patients ,2. IntraThe increase in awareness of IAH and ACS is due to improvements in diagnostic practices and changing treatment paradigms in patients sustaining traumatic injury and those with critical illness ,3. DespiThis review seeks to define IAH and ACS, identify the etiology and presentation of IAH and ACS, identify IAP measurement techniques, identify current management and discuss the implications of IAH and ACS for nursing practice.An integrative review is a method that permits the inclusion of a range of study designs to provide an inclusive evaluation . This prUsing the stated search strategy 514 articles were retrieved. Abstracts were reviewed for relevance to the review aims. Sixty five articles provided information describing the nursing role, the description of the assessment process, diagnosis and management of IAH and ACS Diagnosis of intra abdominal hypertension; (2) etiology of intra-abdominal hypertension; (3) strategies for measuring intra-abdominal pressure; (4) the manifestations of abdominal compartment syndrome; and (5) the importance of nursing assessment, observations and intervention.Intra-abdominal pressure is defined as the pressure created within the abdominal cavity the normal IAP for critically ill adults is 5\u20137\u00a0mmHg ,11. IntrThere are multiple physiological factors that have the potential to alter an individual\u2019s intra-abdominal pressures (IAP). These factors can be categorised as those that are related to;1. A decrease in abdominal wall compliance.2. An increase in intraluminal contents.3. Capillary leakage or fluid resuscitation see Table\u00a0Whilst there are no risk prediction models that will assist in identifying IAH or ACS, elevated peak ventilation pressures, decreased urine output, hypothermia, coagulopathy and acidosis have been described in several studies as the key indicators of an increased mortality -17. ThesMeasurement of IAP is simple, inexpensive, safe and accurate method in determining the presence of IAH. This measurement can guide patient management ,10,18,19Historically physical observation and measurement of abdominal girth were used to determine the presence of IAH. This method of measurement is inaccurate due to a high risk of variability and low inter-rater reliability ,20. A raThe WSACS advocates the use of the modified Kron technique as the gold standard of IAP measurement ,10,11. T1. With the transducer zeroed and positioned in line with the iliac crest and mid-axillar.2. With the patient in a supine position.3. At end-expiration.4. With an instillation volume of no greater than 25\u00a0mL of saline (for bladder technique).5. 30\u201360\u00a0seconds after instillation to allow for bladder detrusor muscle relaxation (for bladder technique) ,16,20-22The reliability of the intermittent measurement guidelines set down by WSACS has been challenged ,23. MoreThere is also a range of opinions regarding the volume of fluid required to be instilled into the bladder to format an accurate pressure reading. Volumes as low as 2\u00a0ml have been used to measure IAP and results are comparable to the use of 25\u00a0ml of normal saline . Fluid vThere are a select few patients in whom IAP measurement via the direct bladder method is not feasible. These include patients with a ruptured bladder, intra bladder hematoma, neurogenic bladder, recent bladder surgery and uro-genital anomalies -33. As sAbdominal compartment syndrome is defined as a sustained IAP greater than 20\u00a0mmHg with a new organ dysfunction or failure regardless of abdominal perfusion pressure (APP) ,12,13,15Abdominal compartment syndrome is further classified into three groups primary, secondary and recurrent ACS.Primary ACS occurs as a result of injury or disease to the abdominal or pelvic region that frequently requires early radiological or surgical intervention or, conditions that develop post abdominal surgery requiring surgical intervention ,7,10,11.Secondary ACS is an often unavoidable progression of the ICU patient\u2019s pathology and refers to conditions that do not originate from the abdominal or pelvic region . SecondaRecurrent ACS is the reoccurrence of ACS after surgical or medical treatment of either primary or secondary IAH or ACS ,10-12,34The abdominal perfusion pressure (APP) has been identified as an indicator for adequate abdominal perfusion ,36. AbdoThere is considerable debate regarding the applicability of absolute IAP ranges in the management of critically ill patients ,6. As suIdentifying patients at risk is the initial step in the recognition and diagnosis of these pathologies . It is eThere are recognised independent risk factors for the development of IAH and ACS See Tab. In addiIn spite of the diverse literature discussing IAH and ACS, there is limited literature specific to the nursing care for patients with IAH or ACS. Patients with IAH or ACS will be most frequently encountered in ICU, high dependency units (HDU), coronary care units (CCU) and emergency departments (ED). Recently, it has been proposed to expand IAP and ACS monitoring beyond traditional critical care areas to enable early detection of the clinical deterioration in in susceptible patients thus improve patient outcomes ,39.The complex presentation of patients with IAH or ACS requires an advanced practice nurse\u2019s clinical expertise and vigilant monitoring is essential ,21. AdvaSpecific nursing management is focused on assessing organ function, pain management, vital signs, perfusion to the lower extremities, assessment of wound drainage and output, ongoing assessment for reoccurrence of IAH or ACS and provision of support to patients and their families ,42,43.Due to the adverse effects of IAH and ACS on patient morbidity and mortality and abdominal compartment syndrome (ACS) occur frequently in critical care and can alter organ perfusion and end organ function.\u25cf\u2002Measurement of Intra-abdominal pressure (IAP) is done via the bladder using the modified Kron technique.\u25cf\u2002Abdominal compartment syndrome (ACS) is classified as an IAP greater than 20\u00a0mmHg with a new organ dysfunction.\u25cf\u2002Critical care nurses play a significant role in the recognition and management of IAH and ACS.The authors declare that they have no competing interests.LH: Study design, data analysis and interpretation, manuscript preparation. SAF: Study design, interpretation of data, manuscript preparation. KH: Study design, interpretation of data, manuscript preparation. PJN: Interpretation of data, manuscript preparation. PMD: Study design, interpretation of data, manuscript preparation. All authors read and approved the final manuscript."} +{"text": "The last several decades have seen many advances in the recognition and prevention of the abdominal compartment syndrome (ACS) and its precursor, intra-abdominal hypertension (IAH). There has also been a relative explosion of knowledge in the critical care, trauma, and surgical populations, and the inception of a society dedicated to its understanding, the World Society of the Abdominal Compartment Syndrome (WSACS). However, there has been almost no recognition or appreciation of the potential presence, influence, and management of intra-abdominal pressure (IAP), IAH, and ACS in pregnancy. This review highlights the importance and relevance of IAP in the critically ill parturient, the current lack of normative IAP values in pregnancy today, along with a review of the potential relationship between IAH and maternal diseases such as preeclampsia-eclampsia and its potential impact on fetal development. Finally, current IAP measurement guidelines are questioned, as they do not take into account the gravid uterus and its mechanical impact on intra-vesicular pressure. Despite the nearing deadline for attaining the World Health Organization's Millennium Development Goals that include improving maternal health worldwide by 2015, the reality remains that over one-half million expectant or new mothers die suddenly and unpredictably ,4. FurthSince the inception of the World Society of the Abdominal Compartment Syndrome (WSACS) in 2004, many advances in the recognition, the treatment, and especially the prevention of the abdominal compartment syndrome (ACS) have occurred ,6. When in extremis [However, despite seminal work early in the twentieth century by an obstetrician, Paramore , there hextremis -13. As pextremis . In the extremis , in whomextremis . The relextremis .2/FiO2 ratio of 53, despite receiving 100% fractional inspired oxygen (FiO2). Her initial lactate was 2.1 mmol/l, hemoglobin was 10.5 g/dl, and creatinine was 58 \u03bcmol/l with a urine output of greater than 30 ml/h. Her physiologic data can be found in Table A 16-year-old female, estimated to be at 32 weeks gestation, was transferred to a tertiary intensive care unit (ICU) from a peripheral hospital. She required intubation and ventilation following a 2-week prodrome of progressive cough. Her ICU admission revealed severe hypoxemia with a PaO2O. The central venous pressure (CVP) increased to 27 mmHg, and severe subcutaneous emphysema developed 'from head to toe'. Transthoracic echocardiography revealed a hyperdynamic, underfilled heart. She continued to deteriorate, prompting a consult to cardiac surgery for extracorporeal life support (ECLS) as a last resort to her spiraling physiologic decline.The patient was ventilated using high-frequency oscillatory ventilation in combination with nitric oxide (NO), but the oxygenation did not improve significantly. Within 15 h of admission, bilateral chest tubes were required for presumed barotrauma-related pneumothoraces. She became anuric in association with a rising creatinine of 122 \u03bcmol/l. Nine hours later, she further decompensated, requiring an increase of the mean airway pressure (MAWP) to 50 cmHObstetrics was re-consulted and noted that sonographic windows during a repeat fetal ultrasound were completely absent. After much debate, it was decided that cannulation for ECLS would precede a caesarean section (CS). In the operating room, the ECLS cannulation procedure proved difficult and prolonged, resulting in iatrogenic injuries to both the groin and subclavian vessels, and transfusion of six units of red blood cells. Finally, after cannulation, the ECLS circulatory flows were inadequate at 1 to 1.5 l/min.2) increased above 90% for the first time since her admission. The ECLS flows only improved partially. The fetus was successfully delivered within 20 min. Upon evacuation of the uterus, there was a distinct improvement in the patient's ventilation and oxygenation. The ECLS flows immediately improved to normal levels (4 to 4.5 l/min); SpO2 was 100%; and the NO and jet ventilator were quickly weaned. There was a spontaneous diuresis of clear urine intraoperatively. The neonate was intubated after delivery, immediately taken to the neonatal intensive care unit, and eventually discharged on day 27 but had multiple hospital admissions thereafter.Thereafter, the CS was performed by a midline laparotomy. Upon incising the peritoneum, an unexpected gush of air was released. There was an immediate improvement in mechanical ventilation; pulse oximetry was determined to be caused by a severe influenza A infection, prompting publication of a plea for routine vaccination of all eligible patients with the influenza vaccine, although no mention of the potential role of ACS was made . The motThe evidence to support the conclusion that ACS significantly contributed to her spiraling decline included the high MAWP of 50 mmHg, a well-described inciting factor for tension pneumoperitoneum -22. The in utero until such time it is ready for a physically independent life. The maternal physiologic changes that occur in pregnancy are multisystemic and far-reaching, not the least of which is the adaptation to accommodate the gravid uterus. On average, the uterus contributes 1 kg to the overall weight gain in pregnancy, while the amniotic fluid, fetus, and placenta comprise approximately 5 kg in additional weight [As IAH/ACS impacts almost all of the body's organ systems, it is important for intensivists to understand the normal alterations in these systems that occur with pregnancy. The pregnant state involves a complex and remarkable interplay of both adaptive and supportive physiology, allowing a fetus to grow and thrive for a finite duration l weight .2), gas exchange, and acid/base balance are all affected by several factors including the growth of the feto-placental unit, progesterone levels, and carbon dioxide production. On average, maternal VO2 increases by 15% to 20% [2 increased with advancing gestation by 28% [To accommodate this growth, the thoracic cage increases in both anteroposterior and transverse diameters . The hor% to 20% , althougn by 28% . Thus, wn by 28% .In addition, there is a 50% increase in plasma volume resulting in dilutional anemia and overall increase in circulating blood volume of 40% ,27. CardKnowledge of normal versus pathologic IAPs for any population in the ICU would seem intuitive for patient care . To dateThe state of the science in this regard is well symbolized by the fact that until very recently, the best evidence concerning IAP in pregnancy was obtained through rectal manometry on primarily primigravid inmates of an institution for 'fallen women' and published in 1913 . More reWe similarly measured the IAP in 20 term parturients under spinal anesthesia . The IAPThus, the above studies highlight currently unresolved issues regarding the necessary trade-offs between the use of a standardized and reproducible reference position to obtain meaningful IAP data, and the reality of patient safety. This is akin to the concern regarding the positioning of ventilated patients fully supine to measure IAP, while increasing aspiration risks ,34,35. LPreeclampsia, part of a spectrum of hypertensive disorders of pregnancy, is defined as the development of arterial hypertension and proteinuria after 20 weeks gestation and is aTwo dramatic case reports described overt ACS as a complication of preeclampsia-eclampsia/HELLP syndromes requiring urgent life-saving interventions ,13. The E) [E decreased significantly with increased age and gravidity [Even as early as the 1900s, investigators had suggested uncompensated elevated IAP as a possible etiologic factor in the development of preeclampsia ,46. WhilE) and evenravidity . Sugermaravidity . He specravidity .That ACS occurs in this patient population is not really the question. Given the evidence in the literature to date Table , it is lin utero is subject to transmitted IAP [2. In a gravid rabbit model, Karnak et al. examined the relationship between maternal IAP and intra-amniotic pressure (IAMNP) through catheters inserted into both the intraperitoneal and intra-amniotic cavities at 20 days of gestation. Intraperitoneal air was insufflated to an IAP of 20 cmH2O. They found that IAMNP was linearly related to IAP as defined by IAMNP = IAP \u00d7 0.8 + 2.0. Further, they found that the elevation of IAMNP to 15.6 cmH2O via the elevation of the IAP (to 17 cmH2O) altered the contractile properties of the fetal bladder [Despite the limited understanding of IAH in maternal care, even less is known regarding its effects on the fetus. Whether there are subclinical effects of even modest elevations of maternal IAP on the fetus is completely unknown. Several animal studies have confirmed that the mammalian fetus tted IAP ,53. IAH tted IAP such as While we are aware of no modern human data correlating maternal IAH with any known effects on the fetus, concerns regarding the fetal-placental unit are neither entirely novel nor implausible. Tanyel hypothesOvarian hyperstimulation syndrome (OHSS) is a not an uncommon complication of ovulation induction for assisted reproduction ,57. The Like OHSS, rapid growth in abdominal girth, dyspnea, abdominal pain, and other overt symptoms of ACS in other gynecological conditions must also be considered in the differential. Patients undergoing ovulation induction are also at increased risk of ovarian torsion and ectopic pregnancy . Meigs' It is currently a recommended standard for any newly admitted critically ill patient with any two IAH risk factors to have baseline IAP measured . The cri2: fractional inspired oxygen; IAH: intra-abdominal hypertension; IAMP: intra-amniotic pressure; IAP: intra-abdominal pressure; ICU: intensive care unit; MAWP: mean airway pressure; OHSS: ovarian hyperstimulation syndrome; SpO2: pulse oximetry; VO2: oxygen consumption; WSACS: World Society of the Abdominal Compartment Syndrome.ACS: abdominal compartment syndrome; CS: caesarean section; CVP: central venous pressure; ECLS: extracorporeal life support; FiODr. Andrew W Kirkpatrick is the principal investigator of an ongoing randomized trial of vacuum therapy in open-abdomen management in critical illness/injury that is funded by the KCI Corporation, although there is no personal benefit to him. He is the Chairman of the Guidelines Committee of the World Society of the Abdominal Compartment Syndrome. Dr. Rosaleen Chun declares that she has no competing interests.RC drafted the original manuscript; was responsible for the conception, design, the data acquisition, analysis and interpretation of this original work; as well as revision for important intellectual content. AWK made substantial contributions to the final manuscript, the data acquisition, analysis, and interpretation of this work; as well as critically revised this original work for important intellectual content. Both authors read and approved the final manuscript."} +{"text": "Autophagy is an intracellular pathway for bulk protein degradation and the removal of damaged organelles by lysosomes. Autophagy was previously thought to be unselective; however, studies have increasingly confirmed that autophagy-mediated protein degradation is highly regulated. Abnormal autophagic protein degradation has been associated with multiple human diseases such as cancer, neurological disability and cardiovascular disease; therefore, further elucidation of protein degradation by autophagy may be beneficial for protein-based clinical therapies. Macroautophagy and chaperone-mediated autophagy (CMA) can both participate in selective protein degradation in mammalian cells, but the process is quite different in each case. Here, we summarize the various types of macroautophagy and CMA involved in determining protein degradation. For this summary, we divide the autophagic protein degradation pathways into four categories: the post-translational modification dependent and independent CMA pathways and the ubiquitin dependent and independent macroautophagy pathways, and describe how some non-canonical pathways and modifications such as phosphorylation, acetylation and arginylation can influence protein degradation by the autophagy lysosome system (ALS). Finally, we comment on why autophagy can serve as either diagnostics or therapeutic targets in different human diseases. Autophagy is an evolutionarily conserved eukaryotic process that can be initiated in response to both external and intracellular factors, including amino acid starvation , growth Unfolded or misfolded cellular proteins are generally degraded by the proteasome; however, proteasome activity can be inhibited by large protein polymers due to their inability to pass through the narrow proteasome channel; thus, autophagy is stimulated to eliminate those protein complexes , 15. MacAutophagy occupies a central position in the maintenance of cellular homeostasis by directing protein degradation, and the process adapts cells to adverse micro-environmental conditions. Accordingly, unraveling more details about autophagic protein degradation pathways may not only improve our understanding of cell metabolism and fate, but may also help us understand a diverse range of human diseases. In this review, we focus on diverse autophagy-mediated protein degradation processes and their regulation.The ubiquitin proteasome system (UPS) and the autophagy-lysosome system (ALS) are alternative ways to categorize protein degradation based on the functions of the proteins being degraded. It is well known that large protein complexes cannot fit into the narrow proteasomal channels and are thus directed to lysosomal disruption , 15.There are three types of proteins in cells that are degraded by autophagy Figure ; the firChaperone-mediated autophagy (CMA) is a type of autophagy that degrades soluble or unfoldable proteins in a molecule-by-molecule fashion Figure . In contHSC70 is a member of the heat shock protein 70 family and is located in the cytosol or the lumen of lysosomes . ProteinThe criterion for the protein to be a putative CMA substrate is the presence of a peptide sequence that is biochemically related to the KFERQ motif. In brief, the motif is flanked by a glutamine (Q) and contains one acidic residue (D or E), a basic residue (K or R), a hydrophobic residue , and a fifth residue that can be basic or hydrophobic but cannot be negatively charged . In partRecent studies have indicated that oxidative stress can activate the CMA pathway and that this potentiation occurs at least partially because oxidized proteins are more easily unfolded to facilitate translocation into the lysosomal lumen. In addition, lysosomes are more active under oxidative stress. For example, the expression of LAMP2A and Lys-HSC70 increased in the lysosomal membrane and lumen during oxidative stress . MoreoveIn addition to oxidative stress and oxidation, other forms of modifications such as phosphorylation, ubiquitination and acetylation can participate in the regulation of CMA. When Phosphoprotein enriched in diabetes (PED) was phosphorylated at Ser104 or Ser116 near the KFERQ-like motifs, its interaction with HSC70 was reduced, thereby precluding its degradation . In contAlthough the precise mechanism by which the modifications affect the interaction between the protein and HSC70 is not yet clear, many CMA substrates were identified during the last decade and were listed in J. Fred Dice's article . In thisAutophagy receptors and the ATG8 family are two primary elements in selective macroautophagic protein degradation. Autophagy receptors bind cargo and the ATG8 family plays a pivotal role in the selective macroautophagy process by promoting the entry of cargo receptors into the autophagy cascade via interaction with the LC3-interacting regions (LIR) of the autophagy receptor , 59. TheTo date, several autophagy receptors that mediate protein degradation have been identified, including TOLLIP, SQSTM1/p62, NDP52, OPTN and NBR1. These receptors contain four different ubiquitin-binding domains: the coupling of ubiquitin conjugation to endoplasmic reticulum-associated degradation domain (CUE), the ubiquitin-associated domain (UBA), the ubiquitin-binding zinc finger domain (UBZ) and the ubiquitin binding in ABIN and NEMO domain (UBAN), along with one or two LIR domains Figure 63, 64], 6463, 6Selective protein degradation by macroautophagy has gained increasing attention due to increased studies of the roles of SQSTM1 in ubiquitinated protein degradation . Recent Similar to UPS, there is strong evidence supporting the recognition of ubiquitinated proteins by receptors, which helps to include them in the macroautophagy cascade Figure 63]. Pr. Pr63]. There are two E1 activating enzymes, 40 E2 conjugating enzymes and more than 500 E3 ligases for ubiquitination in mammals . DistincMoreover, the post-translational modifications of autophagy receptors can influence their interaction with ubiquitinated substrates. The phosphorylation of p62 S403 in its UBA domain by casein kinase 2 (CK2) enhances its binding affinity to ubiquitinated proteins and then promotes the autophagic clearance of the substrate . HoweverUnlike the ubiquitination in the ALS, deubiquitinating enzymes that negatively regulate autophagic protein degradation in mammals have rarely been reported. To date, only one deubiquitinating enzyme, USP36, has been confirmed. USP36 can inhibit selective macroautophagy by preventing the accumulation of ubiquitinated proteins, but the specific substrates of USP36 (whose targeting to autophagosomes is affected) are not yet known . TherefoSubstantial evidence has implicated ubiquitin as the culprit responsible for macroautophagic protein degradation. However, recent results argue against the possibility that ubiquitin is the ubiquitous code in selective macroautophagy Table . For exaIn addition to the direct interaction with an autophagy receptor or ATG8 family member Figure , proteinMacroautophagy and CMA are two distinct mechanisms of protein degradation; however, crosstalk between these pathways has been reported. BAG3 coupled with HSC70, which participates in CMA by recognizing the KFERQ motif in substrates, can release HSC70 substrates to the dynein motor complex, thereby mediating aggresome targeting and the macroautophagic degradation of chaperoned substrates . This prIn addition to the crosstalk between different autophagy pathways, autophagy and proteasome pathways have been demonstrated to work complementarily to degrade proteins under some circumstances. For example, when the proteasome system cannot degrade large protein aggregates, HSP90 and Poh1 help the aggregates dissociate from 20S proteasomes while macroautophagy is activated to facilitate clearance of these protein aggregates . TherefoMoreover, the proteasome and lysosome are believed to degrade misfolded proteins with the help of heat shock proteins, and it was therefore believed that these pathways are both activated to eliminate misfolded proteins that are able to pass through the narrow proteasome channel. Interestingly, the SOD1 G93A mutant was reported to be ubiquitinated by E3 ligase gp78, which targets the protein for proteasome degradation . HainingPrevalent human diseases, including neurodegenerative diseases and carcinomas, have been reported to be attributable to dysfunction of protein degradation; therefore, certain functional elements in ALS or UPS tend to be used as either diagnostic or therapeutic targets in the treatment of these diseases.A recent finding has indicated that lysosomal proteins LAMP1 and LAMP2 and the autophagy protein LC3B in human cerebrospinal fluid (CSF) may be potential novel biomarkers for Alzheimer disease ; howeverin vitro and in vivo models of myeloma [Similar to ALS, abnormal UPS, including its proteasomes and proteases, is an indicator and target of numerous human diseases . The ant myeloma .Proteasome inhibitors such as Bortezomib and DUB inhibitor PR-619 can trigger autophagy, although the mechanisms of these activities are not yet fully understood , 183. InAutophagic degradation has been demonstrated to be selectively and precisely regulated. However, details regarding this type of degradation pathway remain largely unknown. Ubiquitination has been shown to accelerate autophagic protein degradation by interacting with the ubiquitin-binding domains in autophagy receptors. Interestingly, recent studies showed that, compared with non-arginylated BiP and non-acetylated Huntingtin proteins, arginylated and acetylated proteins strongly bind p62 to undergo degradation by autophagy, though the precise mechanisms remain unknown , 93. ThuLong noncoding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides, are a currently popular study topic in research due to their far-ranging functions in regulating protein-protein and protein-RNA interactions , 188. ThEctopic accumulation and distribution of functional proteins are adverse to human health. Although autophagic removal of insoluble toxic proteins or oncogenic proteins could relieve neurological or tumorous symptoms respectively, specific and effective targeting by autophagy drugs is insufficient compared with numerous UPS intervening agents. Therefore, the questions of whether any drugs that can selectively modulate protein and autophagy receptor interactions or whether E3 ligase inhibitors or DUB inhibitors can control the autophagic degradation of certain protein targets remain to be further determined. Further investigation of autophagic protein degradation mechanisms may help elucidate these questions and provide more unique therapeutic strategies to treat human diseases."} +{"text": "The ubiquitin-proteasome system and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. By now we know that these degradation pathways share certain substrates and regulatory molecules and show coordinated and compensatory function. Two ubiquitin-like protein conjugation pathways were discovered that are required for autophagosome biogenesis: the Atg12-Atg5-Atg16 and Atg8 systems. Autophagy has been considered to be essentially a nonselective process, but it turned out to be at least partially selective. Selective substrates of autophagy include damaged mitochondria, intracellular pathogens, and even a subset of cytosolic proteins with the help of ubiquitin-binding autophagic adaptors, such as p62/SQSTM1, NBR1, NDP52, and Optineurin. These proteins selectively recognize autophagic cargo and mediate its engulfment into autophagosomes by binding to the small ubiquitin-like modifiers that belong to the Atg8/LC3 family. Two major pathways accomplish regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal system. The UPS serves as the primary route of degradation for thousands of short-lived proteins and many regulatory proteins and contributes to the degradation of defective proteins . AutophaThe ubiquitin-proteasome pathway plays a crucial role in governing many basic cellular processes, such as normal protein turnover, protein quality control by degrading misfolded and damaged proteins, signal transduction, metabolism, cell death, immune responses, and cell cycle control . Ubiquit\u03b5-amino group of a lysine residue in the target protein and the carboxyl terminus of ubiquitin. Then, it is transferred to a ubiquitin-conjugating enzyme (E2). Finally, E2 associates with ubiquitin-ligases (E3s) which specifically bind the target substrate and attach ubiquitin through its carboxyl terminal glycine to the protein . The exa protein .The high specificity of the UPS system is tightly associated with the E3 enzymes, as they determine which substrate should be ubiquitinylated and hence usually degraded. Whether the attached ubiquitin is a modification signal or a sign for degradation depends on how it is linked to its substrates: conjugation of a single ubiquitin monomer (monoubiquitinylation) or sequential conjugation of several ubiquitin moieties (polyubiquitinylation) of variable length.\u03b5-amino group of a lysine [The ubiquitin chain could be lengthened by the E2 and E3, sometimes with the help of an accessory factor (E4). The carboxyl terminal glycine of the more distal ubiquitin molecule is bound to the previous ubiquitin molecule through an isopeptide bond with an a lysine . If the a lysine .The 26S proteasome is a 2.5\u2009MDa multicatalytic multisubunit protease, which is made up of two subcomplexes: a barrel-shaped core particle and one or two 19S regulatory particle(s) (RP) on one or both ends of the core particle \u201312. The \u03b1-rings and two inner \u03b2-rings, each of which is made up of seven structurally similar \u03b1 and \u03b2 subunits, respectively. The rings form an \u03b11\u20137\u03b21\u20137\u03b21\u20137\u03b11\u20137 structure creating three continuous chambers inside the particle. Only three of the \u03b2-type subunits in each inner ring are catalytically active. They have threonine residues at their N-termini and show N-terminal nucleophile hydrolase activity. Such a \u201cself-compartmentalized\u201d structure keeps the proteolytic active sites separated in the central chamber and allows regulated substrate degradation only. The proteasome is a multicatalytic protease because the \u03b21, \u03b22, and \u03b25 subunits are associated with caspase-like, trypsin-like, and chymotrypsin-like activities, respectively, which are able to cleave amide bonds at the C-terminal side of acidic, basic, and hydrophobic amino-acid residues, respectively.The 20S CP contains two outer The ubiquitin chains are called K6, K11, K27, K29, K33, K48, or K63 chains depending on which of the seven lysine (K) residues is involved in linkage of monomers in the polyubiquitin polymer identified and characterized. They resemble ubiquitin, as for all Ubls whose covalent attachment to other biomolecules has been experimentally demonstrated, the C-terminal residue is a glycine, and the carboxyl group of this glycine is the site of attachment to substrates . On subs segment .Autophagy is another degradative pathway that occurs in all eukaryotic cells. It is the main system for the degradation of bulk cytoplasmic components in the cell, and it is induced by nutrient starvation for example. Autophagy is crucial for homeostasis in the cell, as it recycles proteins and organelles. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and acting in various aspects of immunity, including the elimination of invading microbes and its participation in antigen presentation. Macroautophagy is the best characterized type of autophagy. In this case the cell forms a double-membrane sequestering compartment called the phagophore, which develops into an autophagosome. After fusion with lysosomes, the content of the resulting autolysosome is degraded and the newly generated monomers are released back into the cytosol for reuse , 17 Fig. Drosophila and mammalian cells [There are 38 known autophagy-related (Atg) genes regulating the steps of autophagosome formation and breakdown. These were identified in yeast genetic screens but they are evolutionarily well conserved also in plants and animals, includingan cells , 19. Inian cells .The transmembrane protein Atg9 and regulators of its trafficking (Atg2 and Atg18) play a role in membrane delivery to the expanding phagophore after the assembly of the Atg1 complex at the single phagophore assembly site (PAS), which is marked by the selective cargo proaminopeptidase I aggregate in yeast. Nucleation of the phagophore at the PAS is controlled by the phosphatidylinositol-3-kinase (PI3\u2009K) complex . Finally, there are two Ubl conjugation systems: the Atg12 and Atg8 pathways which are responsible for vesicle expansion Figure .Autophagosomes undergo a maturation process in animal cells, which involves the recruitment of the SNARE protein syntaxin 17 \u201324. InteMacroautophagy has long been considered as a nonselective process responsible for bulk degradation of cytoplasmic components. The autophagy pathway appeared during evolution as an adaptation mechanism of the eukaryotic cell to starvation, allowing mobilization of nutrients in the cell by forfeit materials of the cytosol. Additionally, it became indispensable for specific degradation of unnecessary or toxic structures: proteins, organelles, and intracellular pathogens . In contAutophagy requires the Ubls Atg12 and Atg8/LC3 Figures . Atg12, Atg8, the other Ubl regulator of autophagy, is expressed with a C-terminal arginine residue in yeast, which is removed by the cysteine protease Atg4 leaving a glycine residue at the C-terminus . BiochemIn the last decade, emerging evidence revealed that autophagy can distinguish and direct specific cargos to the lysosome. Different terms were coined to distinguish between different targets. The most investigated processes are mitophagy: the selective removal of defective or excess mitochondria ; aggrephA molecule convenient to link a process with its substrate needs to carry at least two distinct functional domains: one that recognizes the target and another that transports it to the site of operation. How does it work in the case of selective autophagy? The best known mechanism to solve the problem of distinction between the different cytoplasmic components deemed for engulfment is to bring properly marked cargos to the inner surface of the growing phagophore. Accordingly, the precise delivery is generally ensured by interaction of the adaptor both with the membrane-anchored form of Atg8/LC3 and the main targets that are usually polyubiquitinylated . Drosophila experiments. They showed that the loss of basal autophagy in the brain resulted in large-scale accumulation of ubiquitinylated proteins [The first clues for the role of protein ubiquitinylation as a signal for selective autophagy came from Atg knockout mice and someproteins \u201340. Drosophila revealed that p62 is required for the aggregation of ubiquitinylated proteins and thus plays essential roles for their autophagic clearance [Recognition of ubiquitinylated proteins during autophagy is mediated by ubiquitin receptors interacting with ubiquitin noncovalently, via their ubiquitin-binding domains. p62/SQSMT1 (hereafter p62), the first protein reported to have such an adaptor function , was orilearance , 45. Thelearance , 47. p62learance , 49. NBR1, which, via its own PB1 domain, is able to interact with p62, and through its own UBA domain and LIR it can participate in the recruitment and autophagosomal degradation of ubiquitinylated proteins [Another adaptor used in selective autophagy is the above-mentionedproteins . In planproteins , 52.Optineurin and NDP52 have been recently described as xenophagy receptors, utilizing the autophagic machinery for restriction of ubiquitinylated intracellular pathogens [\u03baB signaling [athogens . Both ofathogens , 55 and ignaling , 57.While these receptors all mediate degradation of ubiquitinylated cargos, there are other more specific adaptors acting on removal of damaged or surplus mitochondria or peroxisomes (such as Atg30 and Atg36). They recognize particular binding partners on the surface of their target organelle and, through their LIR sequence, ensure their delivery to the maturing autophagosome , 59. It As individual p62-ubiquitin interactions are rather weak, the starting point of the polyubiquitinylated aggregate formation is presumably the p62 self-oligomerization via its PB1 domain . However Drosophila cells, which is very similar to the location of PAS near the vacuole/lysosome in yeast [Moreover, it was recently reported that phagophores may preferentially form at p62 aggregates near lysosomes inin yeast , 65. It in yeast . MTORC1 in yeast \u201369. Thesin yeast , 71. Drosophila fat body cells [The role of p62 in the regulation of autophagy is controversial. It was suggested to promote MTORC1 activation by contributing to its translocation to the lysosomal surface. Therefore, p62 reduction, similarly to MTORC1 inactivation, may activate autophagy . Howeverdy cells . Thus, tAs p62 can shuttle between the nucleus and the cytoplasm (in the nucleus it is thought to recruit proteasomes to nuclear polyubiquitinylated protein aggregates), it can even export ubiquitinylated substrates from the nucleus into the cytosol, where autophagy offers a more robust degradative capacity . de novo p62 protein synthesis by providing autophagy-derived amino acids [Since p62 itself is removed from the cytoplasm mainly by autophagy, its amount is generally considered to inversely correlate with autophagic activity , 47. Accno acids .\u03b1-activated cells. The complex including the RIP kinase, atypical PKCs and TRAF6, and a K63 ubiquitin ligase plays a critical role in the phosphorylation of IKK\u03b2 leading to activation of the NF-\u03baB transcription factor [\u03b2 production: p62 was found to bind the JNK and ERK kinases, hence further increasing NF-\u03baB activation and, as a consequence, pro-IL-1\u03b2 expression. In addition, p62 accumulation was found to promote caspase-1 activation in inflammasomes, which is required for IL-1\u03b2 proteolytic processing [\u03b2 was significantly enhanced due to elevated caspase-1 activity in their macrophages [p62 was originally described as a scaffold protein ensuring the formation of signaling hubs, since, through different binding domains, it can establish interactions with many types of enzymes. As a consequence, it is able to integrate signaling routes involving particular kinases and ubiquitin-mediated pathways . This wan factor . Enhanceocessing . Interesrophages .\u03baB-mediated neuronal survival and differentiation in response to NGF [\u03baB signalization [\u03baB connection has a role in tumorigenesis as well, since p62 is necessary to NF-\u03baB-dependent survival in Ras-transformed cells [p62, likewise in association with TRAF6 and aPKCs, is needed for the NF-e to NGF and alsoe to NGF . p62 mutlization . The p62ed cells .\u03baB signaling pathway. In human monocytes, high level of inflammation due to autophagy impairment is associated with p62 accumulation and the consequent overactivation of the NF-\u03baB pathway [\u03baB signalization may be regulated directly by the rate of NF-\u03baB removal. Targeted degradation of the p62-NF-\u03baB p65 subunit complex by p62-mediated selective autophagy may play a key role in bone marrow derived macrophage differentiation [The autophagy adaptor function of p62 also has an impact on the NF- pathway . In acco pathway , a previ pathway . In addintiation .The important role of p62 in innate immunity does not only rely on regulation of immune signaling responses. As an autophagy adaptor, p62 takes part in the elimination of ubiquitinylated intracellular pathogens; some infecting agents even target this step to escape from the defensive system of the cell. The coxsackievirus B3, through the activity of one of its proteases, cleaves p62 which results in impairment of selective autophagy and host defense . Moreovep62 is also involved in the regulation of apoptosis. p62-mediated aggregation is needed for the activation of polyubiquitinated caspase-8 . It was Another well-known signaling pathway influenced by p62 is the oxidative stress response, which is regulated by the Keap1-Nrf2 system. Through its KIR motif , p62 is Ubiquitin and ubiquitin-like proteins (Ubl) share functional similarity. The different Ubls are activated and conjugated to substrates by similar biochemical mechanisms.Ubiquitinylation is frequently needed for substrate recognition and renders selectivity to autophagy in eukaryotes.The connection between ubiquitinylation and autophagy is provided by autophagic adaptor proteins (or autophagy receptors), which bind both ubiquitin and autophagy specific Ubl modifiers like Atg8/LC3 family proteins.Atg8/LC3 is required for the biogenesis of autophagosomal membrane and also mediates selective autophagy via the recruitment of LIR-containing autophagy receptors that recognize and select cargo.Autophagy receptors such as p62 regulate the selective autophagosomal degradation of large protein aggregates, mitochondria, and bacterial pathogens.p62 may play an important role also as a regulator of autophagy; moreover, it may even be involved in the formation of the autophagosome.As a scaffold protein, p62 operates in signaling pathways which, through the link provided by p62, can also be regulated by selective autophagy."} +{"text": "Many progressive neurodegenerative diseases, including Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and frontotemporal lobe dementia, are associated with the formation of insoluble intracellular proteinaceous inclusions. It is therefore imperative to understand the factors that regulate normal, as well as abnormal, protein recycling in neurons. Dysfunction of the ubiquitin-proteasome or autophagy pathways might contribute to the pathology of various neurodegenerative diseases. Induction of these pathways may offer a rational therapeutic strategy for a number of these diseases. A common theme in neurodegeneration is the age-related accumulation of specific toxic protein species secondary to mutations or misfolding or both. Such disease-related proteins are prone to aggregation, and their oligomers cause toxicity via a gain-of-function mechanism . The remNeurons adapt to cellular stresses by maintaining the integrity of their proteome Figure\u00a0. This maLong-term cellular stress can cause high levels of protein accumulation over time and this can lead to chaperone depletion and compromise the refolding of these accumulated proteins. When refolding is not sufficient to maintain cellular homeostasis in these conditions, misfolded proteins are transferred to the degradation pathways ,16. ThesThe UPS is the primary intracellular proteolytic system responsible for the maintenance of rapid protein turnover in the cytosol and nuclei of cells, including the selective removal of abnormal and misfolded proteins. This degradation occurs in a two-step process: (a) the targeting of proteins through the covalent linkage of specific branched polyubiquitin chains and (b) the degradation of the tagged proteins by the downstream 26S proteasome complex. The 26S proteasome is composed of two sub-complexes: the 19S regulatory particle and the 20S core particle. In neuronal cells, dynamic control of protein stability is crucial for synaptic development and function, and the UPS plays an important role in the regulation of synaptic proteins . AggregaAutophagy represents an alternate pathway to the UPS for degradation of cytosolic proteins and organelles, including lipid structures and glycoproteins. Autophagy has a role in many biological processes, including nutrient recycling for prolonged survival during starvation and cytoprotection through degradation of insoluble inclusions in various neurodegenerative disorders. Since neurons are post-mitotic cells with polarized morphology and active protein trafficking, they are highly dependent on autophagy for their survival ,4. FurthAutophagy can be subdivided into the macroautophagy-lysosomal system (MALS), chaperone-mediated autophagy (CMA), and mitophagy. MALS is responsible for bulk lysosomal degradation of cytosolic proteins, including protein aggregates and organelles. Mitophagy is the selective degradation of mitochondria. Unlike other types of autophagy, which rely on vesicle-mediated delivery of substrates to the lysosome, CMA is a selective process that involves direct translocation of substrates across the lysosomal membrane. When MALS is induced, a double-membrane compartment called an autophagosome sequesters an area of the cytoplasm and eventually fuses with the lysosome, where lysosomal enzymes degrade its contents. The MALS pathway has been implicated in several neurodegenerative diseases. As neurological disease progresses, there is an accumulation of autophagosomes or autophagic vacuoles in the diseased brains. In general, neurodegeneration is associated with defects at various steps in the autophagic process: (a) the disruption of autophagosome formation, (b) the disruption of autophagosome maturation, and (c) the disruption of autophagosome clearance.Several adaptor proteins or shuttling factors are involved in the transport of protein substrates. Such proteins usually possess ubiquitin-associated (UBA) and ubiquitin-like (UBL) domains and include the ubiquilins (UBQLN) and SQSTM1/p62 . They biWe will now discuss the involvement of protein recycling pathways in the most common age-related neurodegenerative diseases, focusing on the UPS, MALS, and mitophagy pathways Figure\u00a0. CMA has+1) has been found to accumulate in the dystrophic neurites and neurofibrillary tangles of AD [+1 are degraded by the UPS, high levels of UBB+1 are incompletely degraded by the UPS, resulting in inhibition [+1 is mediated by E2-25\u00a0K, which is essential for A\u03b2 toxicity in animal AD models [Alzheimer disease (AD) is the most common cause of senile dementia and is characterized by progressive dementia accompanied by personality changes, psychosis, and language problems. Neuropathology is characterized mainly by extracellular senile plaques, consisting primarily of \u03b2-amyloid (A\u03b2), and intracellular neurofibrillary tangles, with hyperphosphorylated microtubule associated protein tau as a main constituent. Dystrophic neurites surrounding the plaques are ubiquitinated and consist of several ubiquitin-binding proteins, such as UBQLNs and SQSTM1/p62. There is not much evidence that the UPS is involved in degradation of A\u03b2 or its precursor, the \u03b2-amyloid precursor protein (\u03b2APP). On the other hand, tau is inefficiently degraded by the UPS, although the non-canonical 20S proteasomal degradation rather than the ubiquitin-dependent 26S proteasomal degradation seems to have a predominant role . Despitees of AD as well es of AD . Althoughibition . It has D models . There iD models .+)-ATPase and targeting to the lysosome. Several groups have supported these findings under presenilin 1 loss-of-function conditions [Abundant autophagic vacuoles were first identified by electron microscopy studies on AD brain and latenditions -46. Howenditions ,48. Relinditions . Interesnditions .There is some evidence supporting a potential beneficial effect of inducing autophagy with rapamycin in AD . For insin vivo while, with increased \u03b1-synuclein burden, MALS is recruited [Parkinson disease (PD) is a progressive neurodegenerative disorder caused by a selective death of dopaminergic neurons in the substantia nigra and results in a resting tremor, rigidity, slowness of movement, and postural instability. The characteristic pathology of PD includes cytoplasmic inclusions called Lewy bodies, whose major constituent is \u03b1-synuclein. It has been reported that the UPS is the main degradation pathway for \u03b1-synuclein under normal conditions ecruited . It has ecruited . Providiecruited reportedecruited . Mutatioecruited ,60, and ecruited . Parkin Autophagy has a vital role to play in the pathogenesis of PD. Conditional deletion of the autophagy-related protein, Atg7, in the substantia nigra dopaminergic neurons recapitulates many of the pathologic features of PD . MoreoveIt is known that damaged mitochondria accumulate with normal aging. Mitochondrial dysfunction has also been implicated in the pathogenesis of PD. It has been shown that parkin is selectively recruited to damaged mitochondria and is important in their selective elimination . MoreoveMutations in genes encoding lysosomal proteins, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2), have also been linked to PD . GBA mutAmyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease and is caused by selective degeneration of motor neurons in the brain and spinal cord. Progressive weakness leads to paralysis that is ultimately fatal, in most cases, within 5\u00a0years of symptom onset. About 5\u00a0% of patients with ALS develop features of overt and disabling dementia, usually of the frontotemporal lobar type (FTD) . The prein vitro[Direct etiological evidence for the involvement of UPS and MALS pathways was provided by the identification of mutations in UBQLN2 and SQSTM1/p62 in ALS and FTD ,88,89. PAlthough a defective UPS has been suggested to produce ALS-associated protein aggregates, recent studies have revealed a prominent role for autophagy . SurviviAn autophagy defect has also been suggested by genetic studies of ALS and FTD. For instance, mutations in UBQLN2 and SQSTM1/p62 have been reported in ALS and FTD ,89,99. UThe ALS-FTD-associated protein TDP43 is degraded by both the UPS and autophagy pathways, and it has been shown that overexpression of SQSTM1/p62 reduces TDP43 aggregation in an autophagy- and proteasome-dependent manner -116. DefHuntington disease (HD) is caused by a polyglutamine repeat expansion in the huntingtin (htt) gene, resulting in protein aggregation and causing a syndrome of involuntary movements and dementia. Although it is clear that the proteasome is involved in the degradation of mutant htt, the role of proteasomes remains contradictory . InteresBecause of their post-mitotic nature, polarized morphology and complex arborization, neurons are particularly sensitive to alterations in protein homeostasis. A plethora of evidence has firmly established the essential role for the UPS and autophagy pathways in both normal physiological functioning as well as pathophysiological conditions of the nervous system.Impairments in the quality control of proteins and organelles in the neuronal soma, axon, and synapses led to an aggregation of toxic proteins and, thereby, cause neuronal dysfunction and neurodegeneration in diseases such as AD, PD, ALS, FTD, and HD.As reviewed above, several independent studies have shown that autophagy induction can be beneficial as a disease-modifying treatment in experimental models of various neurodegenerative diseases. Of note, rapamycin has been in clinical practice for several years. But as a disease-modifying therapy in neurodegeneration, long-term use of rapamycin may have adverse effects on cognition by dysregulating physiological mTOR signaling, which is important for axonal growth, dendritic arborization, and synaptic plasticity ,131. HowMechanistic correlates for abnormal UPS and autophagy in these diseases have yet to be fully understood. Since the cargo destined for recycling may need to be transported over great distances in a neuron, it is likely that complex processes involving abnormal protein-protein interactions or impaired trafficking (or both) may be involved. A better understanding of these mechanisms will aid in the design of rational therapies for neurodegenerative diseases.AD: Alzheimer disease; ALS: amyotrophic lateral sclerosis; ATP13A2: lysosomal type 5 P-type ATPase; A\u03b2: \u03b2-amyloid; CMA: chaperone-mediated autophagy; FTD: frontotemporal lobar dementia; GBA: glucocerebrosidase; HD: Huntington disease; htt: huntingtin; MALS: macroautophagy-lysosomal system; mTOR: mammalian target of rapamycin; PD: Parkinson disease; UBA: ubiquitin-associated; UBB+1: frame-shift mutant of ubiquitin B; UBL: ubiquitin-like; UBQLN: ubiquilins; UPS: ubiquitin-proteasome system; \u03b2APP: \u03b2-amyloid precursor protein.The authors declare that they have no competing interests."} +{"text": "Failure to adhere to standard item-writing guidelines may render examination questions easier or more difficult than intended. Item complexity describes the cognitive skill level required to obtain a correct answer. Higher cognitive examination items promote critical thinking and are recommended to prepare students for clinical training. This study evaluated faculty-authored examinations to determine the impact of item-writing flaws and item complexity on the difficulty and discrimination value of examination items used to assess third year veterinary students.The impact of item-writing flaws and item complexity (cognitive level I-V) on examination item difficulty and discrimination value was evaluated on 1925 examination items prepared by clinical faculty for third year veterinary students.The mean (\u00b1 SE) percent correct (83.3\u00a0%\u2009\u00b1\u200917.5) was consistent with target values in professional education, and the mean discrimination index (0.18\u2009\u00b1\u20090.17) was slightly lower than recommended (0.20). More than one item-writing flaw was identified in 37.3\u00a0% of questions. The most common item-writing flaws were awkward stem structure, implausible distractors, longest response is correct, and responses are series of true-false statements. Higher cognitive skills (complexity level III-IV) were required to correctly answer 38.4\u00a0% of examination items. As item complexity increased, item difficulty and discrimination values increased. The probability of writing discriminating, difficult examination items decreased when implausible distractors and all of the above were used, and increased if the distractors were comprised of a series of true/false statements. Items with four distractors were not more difficult or discriminating than items with three distractors.Preparation of examination questions targeting higher cognitive levels will increase the likelihood of constructing discriminating items. Use of implausible distractors to complete a five-option multiple choice question does not strengthen the discrimination value.The online version of this article (doi:10.1186/s12909-016-0773-3) contains supplementary material, which is available to authorized users. The goal of examination item preparation is to develop testing methods that do not confuse students and yield scores that accurately reflect the extent to which students have obtained a satisfactory working knowledge of the content. Well-constructed multiple choice examinations represent a versatile assessment tool with the potential to assess students for sufficient working knowledge of the tested content \u20134. DiscrLongest option is correct answer.Grammatical clues or inconsistencies between stem and distractors.*Implausible distractors.*Mutually exclusive distractors.*Use of absolute terms .Use of all of the above.The following standard item-writing flaws are considered to make examination items easier than intended, favoring test-wise students :Longest *Examples appear in Additional file longest option is correct answer is a common mistake made by novice and experienced examination writers in an effort to ensure the correct response is indisputable [Grammatical clues may come in the form of syntax inconsistencies between the stem and incorrect responses (distractors), or may occur if a key word in the stem is repeated in the correct response [Implausible distracters are used to create item uniformity when three or four plausible distracters are immediately apparent to the author [mutually exclusive distractors and conclude that one of the two mutually-exclusive responses is correct, eliminating other options [absolute terms usually render a statement false.The sputable . Grammatresponse . Implause author , 17, 18.all of the above\u201d as the correct response, they need only identify two correct answers among the choices. To determine \u201call of the above\u201d is incorrect, students need only identify one false statement [For students to identify \u201ctatement . High sctatement .Author selection for denotation of the correct response is reported to be statistically predictable. Novice examination writers under-utilize options A and E as correct responses, and overuse option C as the correct response . In one Awkward stem structure. *Extraneous or misleading information in the stem.*Negative stem. Response options are a series of true/false statements.*Use of none of the above.Complex or K-type items. (e.g. A and C)Vague or generalizing terms. .Unfocused question. (Distractors are unrelated or distantly related to a single learning objective.)*The following item-writing flaws may render questions unnecessarily complex and prevent prepared students from demonstrating mastery of the material: , 13, 16.*Examples in Additional file awkward stem structure require students to place each response option in the blank or at the end of the sentence, which can result in an error unrelated to the learning objective. Extraneous or misleading information in the item stem distracts students from the learning objective, lengthens the examination unnecessarily, and decreases the reliability and the validity of the resulting test scores [Item-writing experts recommend that the stem be a complete sentence and represent a stand-alone problem . In othet scores .Negatively-worded multiple choice stems instruct students to identify the incorrect answer among response options. Negatively worded questions are easier to construct than positively worded questions, however, learning objectives are more effectively assessed when students identify a correct answer rather than an incorrect answer [t answer , 16. A st answer .series of true/false statements. (e.g. Which of the following statements is correct/incorrect?) Consequently, students are responding to four or five true/false questions with all-or-none grading, rather than one multiple choice item [One variant of multiple-choice questions requires students to evaluate response options that are essentially a ice item , 21. Wheice item .Use of \u201cnone of the above\u201d does not require students to demonstrate knowledge of the correct answer. When used as a distractor, it typically does not appear plausible to experienced students, and becomes a filler or implausible distractor. Use of \u201cnone of the above\u201d decreases item discrimination and test score reliability . CurrentK-Type questions, also known as complex items, require students to select combinations of individual response options. Response options are typically a series of true/false statements, followed by options such as \u201cA and B\u201d or \u201ctwo of the above\u201d [e above\u201d . K-type e above\u201d , 16, 20.Vague or generalizing terms open examination questions to interpretation. The terms \u201cfrequently\u201d, \u201coften\u201d \u201csometimes\u201d or \u201coccasionally\u201d may hold different meaning to the writer and student, and the impact may vary with circumstances or disease condition [unfocused stem is a broad, open-ended question that does not pose a specific problem and is followed by a series of unrelated response options. This item type is popular because it allows examiners to test a broad range of material, but does not provide an assessment of a specific learning objective, and has a detrimental impact on student and item performance [ondition . An unfoformance , 21.True/false questions tend to be easy to write and efficiently answered. Students can respond to approximately 50 true/false items in the time it takes to answer 30 multiple-choice items , 21. ConExamination item complexity is categorized using a five level scale based on Bloom\u2019s Taxonomy , 25: knoI.Factual recall: Which of the following species does not have a gall bladder?II.Conceptual recall: Which of the following conditions is an example of ventilation-perfusion mismatch?Recall (lower level cognition)III.Direct Application: Simple case scenario - Which of the following diagnostic tests is indicated?IV.Analyze conceptual knowledge: Interpret blood work, radiograph, or complex case scenario.V.Evaluate case management: Case vignette - Which steps were unnecessary?Applied (higher level cognition)Examination items testing lower cognitive thinking (I and II) are easier to write, yet are more prone to item-writing flaws and poor discrimination ability . ChallenThe discrimination index indicates the extent that success on the item is related to the success on the test as a whole, and provides feedback to the examiner regarding item difficulty. The index is the difference between the percentage of correct responses from the upper and lower scores of the class, demonstrating the impact of an item to distinguish between high scorers and low scorers on an examination. Varying values are used to define upper and lower student groups, in many cases, upper and lower quartiles are used. The target value for the discrimination index should be approximately 0.20 for examination items, except intentionally easy or difficult questions , 28\u201330. The goals of this investigation are to determine the impact of item-writing flaws and item complexity on the difficulty and discrimination value of examination items authored by in-house veterinary faculty and administered to third year veterinary students. Most reports of item-writing flaws reflect evaluation of students in undergraduate course work or basic science curriculum , 13, 17,Data were collected from all examination questions administered over the academic year for third year veterinary students . At the start of the investigated academic year, third year student respondents had completed an average of 7.2\u00a0years of post-secondary education. Most veterinary students had completed a baccalaureate degree prior to matriculation. All examination questions were authored by college of veterinary medicine (CVM) faculty members, intended to have one correct response, and assessed via automated grading , question number (order of examination questions), length of stem, length of responses, use of ancillary materials , interpretation of laboratory values, and calculation requirement (yes/no).Two item raters (BRR and DCR) evaluated each examination item independently for item-writing flaws and item complexity. When disagreement was observed between raters, raters discussed and reached consensus for each examination item. Raters had content-area expertise, experience preparing multiple choice items, and NBME item-writing training.Raters evaluated examination items for case-based question format (yes/no) and the presence of the following item-writing flaws: longest response is correct, grammatical clues, implausible distractors, mutually exclusive distractors, use of absolute terms, use of all of the above, awkward stem, misleading/extraneous stem, negative stem, true/false distractors, use of none of the above, K-type responses, use of vague terms, and unfocused question.A rating of item complexity (cognitive level I-V) was assigned by the raters for each question based on modified Bloom\u2019s taxonomy , 3, 26. Data were prepared for analysis by removing questions where more than one response option was deemed correct , questions in which no correct answer was identified , and one question from an instructor with only a single question in the data set.P\u2009<\u20090.05) effects.Main outcome variables (% correct and discrimination index) were log-transformed to normalize distributions. Categorical variables evaluated questions that were difficult with poor discrimination , easy with poor discrimination , and challenging with strong discrimination (< 85\u00a0% correct and index\u2009>0.20). Stepwise regression models were created to evaluate relationships among the percent correct, index values, and categorical description of discriminatory value of question compared with all available variables including course, course type (elective/core), test number within course, instructor, stem length, distractor length, case-based (yes/no), complexity, ancillary (yes/no), and the standard item-writing flaws: longest response is correct, grammatical clues, implausible distractor, mutually exclusive distractors, use of absolute terms, use of all of the above, awkward stem, misleading/extraneous stem, negative stem, true/false distractors, use of none of the above, k-type responses, use of vague terms, and unfocused question. To account for lack of independence of questions, course, test number within course, and instructor were forced into all models. Multivariable models were created using minimum Bayesian information criteria to generate a final model including only significant representing 39 credit hours (33 core and six elective credits); 1689 questions were multiple choice items and 236 were true/false items.n\u2009=\u2009554) were identified as free of item-writing flaws. One item-writing flaw was noted in 33.9\u00a0% of the questions, and 37.3\u00a0% were identified to have more than one item-writing flaw . The frequency of specific item-writing flaws appears in Table\u00a0Approximately 28.8\u00a0% of examination items discrimination index . Author bias was not detected in the placement of correct response options with four response options or five response options .Of 236 true/false items, 111 were true (true was the correct response) and 126 were false statements . The mean (\u00b1 standard error) percent correct of true statement items was higher (95.2\u00a0%\u2009\u00b1\u20096.7) and the mean discrimination index was lower (0.068\u2009\u00b1\u20090.05) than the mean percent correct (92.3\u00a0%\u2009\u00b1\u20097.9) and discrimination index (0.108\u2009\u00b1\u20090.080) for false statement examination items.More than half of all examination items (61.6\u00a0%) were considered lower level recall questions with 401 items (20.8\u00a0%) categorized as level I factual recall and 785 items (40.8\u00a0%) categorized as level II conceptual recall. Six hundred and four questions (31.4\u00a0%) required direct application of knowledge in a simple scenario (Level III) and 133 examination items (6.9\u00a0%) required students to analyze conceptual knowledge through interpretation of visual aids and/or complex case material (Level IV). Two examination items asked students to evaluate procedural knowledge through the presentation of a case vignette (Level V). These two items had the longest stem length in this series of 1925 items (12 and 17 lines), highlighting the limitation of this question type.n\u2009=\u200981), followed by line drawings or ECG tracings (n\u2009=\u200922), radiographic or ultrasonographic images (n\u2009=\u200914), and video recordings (n\u2009=\u20096). Forty-nine examination questions required the examinee to perform a calculation, and 60 questions required students to interpret four or more laboratory values. Six hundred and seventy three examination items (35\u00a0%) were classified as case-based questions. The majority of case-based questions (80.1\u00a0%) and items requiring interpretation of ancillary materials (72.2\u00a0%) were classified at Level III complexity or higher.Interpretation of ancillary materials within examination items was uncommon (12\u00a0%). Only 123 examination items (6.4\u00a0%) required interpretation of a visual aid; gross or histologic images were most common percent correct was 83.3\u00a0% \u00b1 17.5) and the mean discrimination index was 0.18 (\u00b1 0.17). As question complexity increased (cognitive level I-IV), the percentage of correct responses decreased of examination items were classified as difficult, discriminating questions . Multivariable analysis revealed four question characteristics were associated with the likelihood of creating difficult, discriminating examination items: item complexity, series of true/false distracters, implausible distractors, and all of the above. Not surprisingly, as item complexity increased, the likelihood of creating a discriminatory question increased of examination items were categorized as poorly discriminating, easy questions . Multivariable analysis revealed item complexity, presence of implausible distractors, and series of true false distractors were associated with the likelihood of creating a poorly discriminating, easy question. As question complexity increased (cognitive levels I-IV), the likelihood of creating an easy question decreased of examination items were categorized as poorly discriminating, difficult questions . These parameters were selected to identify characteristics of questions that were unproductive in the examination process. Due to the sparse data in this category, statistical models did not converge.Only 3.4\u00a0% was consistent with target values for examination average in professional education, and the discrimination index (0.18) was disappointingly lower than recommended (0.20) , 30. TheExamination items with implausible distractors were less able to discriminate prepared from unprepared students (DI ~0.09), and were easier for all students (~95\u00a0% correct) than other examination items. Not surprisingly, the probability of writing challenging, yet discriminating questions decreased when implausible distractors were used. The negative impact of implausible distractors on indices of item quality is a recurring theme in medical education .Implausible distractors are often used as filler, because it is difficult to develop three or four plausible distractors . InstrucExamination items with a series of true/false response options were identified more frequently (~11\u00a0%) in these data than reported by others (2.8\u00a0%) in medical education . The curHigher cognition items require students to assimilate facts, apply knowledge, and predict outcomes, and are more discriminating than factual recall. As question complexity increased (cognitive level I through IV), examination items became more challenging (lower percent correct) and more discriminating; higher question complexity was identified as a feature of difficult, discriminating questions. Medical professionals are expected to use clinical findings and diagnostic results to make decisions that guide treatment planning and prognosis , 6, 26. Item-writing experts concede it is difficult and time-consuming to develop multiple-choice items that measure higher cognitive skills , 29, 38.In the current study, the discrimination index of true/false questions was poor overall. However, false statements were slightly more discriminating than true statements. Discriminating true/false examination items are difficult to construct. Student guessing negatively impacts the discrimination value of true/false questions. Historically, false statements are slightly more discriminating than true statements, and experts recommend test-writers provide slightly more false statements than true statements for that reason , 20. As If unsure of the correct response, students are coached to select option C and avoid option E to increase their chance of obtaining a higher score. In the current study, correct answers were distributed across all options ; no author bias was noted in the placement of correct options. Most CVM faculty members are aware of these patterns and randomize response options alphabetically or numerically. Examination authors are encouraged to evenly distribute correct responses throughout all response options.There are two major limitations of this investigation. One is the source of analyzed examination items. All examination items were written by clinical faculty, intended to test information delivered via didactic course work during the third year of the veterinary curriculum from a single institution. The results may not directly extrapolate to course work in basic sciences, other veterinary institutions, or other medical disciplines. The second is the interpretation of Bloom\u2019s taxonomy. Other investigations in clinical medicine employed alternative modifications of Bloom\u2019s taxonomy with fewer categories . ExtrapoAlthough many item-writing flaws identified in this study did not impact the indices of difficulty or discrimination value, standard item-writing guidelines should be followed to improve the clarity and consistency of examination items. Item-writing flaws identified as disruptive to indices of performance for professional students include implausible distractors, use of \u201call of the above\u201d, and series of true/false response options. Faculty training should place particular emphasis on avoiding these item-writing flaws.Higher question complexity (cognitive level III through IV) was identified as a feature of discriminating examination questions. Examination items that require higher cognitive skills are correlated to student learning and development, particularly in preparation for clinical training , 27, 38."} +{"text": "Harmonia axyridis (Coleoptera: Coccinellidae) makes better use of information from a plant-prey (Vicia faba - Aphis fabae) system compared to the native Oenopia conglobata. Y-tube olfactometer bioassays revealed that both species used olfactory cues from the system, but H. axyridis exhibited a more complete response. This species was also attracted by plants previously infested by aphids, indicating the capacity to exploit volatile synomones induced in plants by aphid attack. Oocyte resorption was investigated when different olfactory stimuli were provided under prey shortage and the readiness of new oogenesis was measured when prey was available again. H. axyridis exhibited higher plasticity in oogenesis related to the presence/absence of plant-aphid volatiles. Our results support the hypothesis that H. axyridis is more reactive than O. conglobata to olfactory cues from the plant-prey system.Understanding the traits that might be linked with biological invasions represents a great challenge for preventing non-target effects on local biodiversity. In predatory insects, the ability to exploit habitats for oviposition and the physiological response to prey availability differs between species. Those species that respond more readily to environmental changes may confer to their offspring a competitive advantage over other species. Here, we tested the hypothesis that the invasive It has been estimated that from 1906 to 1991, 79 exotic species caused $97 billion damage to the economy of the United States2.With increased globalization, several exotic species are introduced into new areas every year, either accidentally or intentionally. Some species do not find suitable conditions, while others successfully establish and, as in the case of natural enemies that are introduced within accurate biological control programs, show beneficial effects on local biodiversity, economic activities and the environment. However, many non-native species, because they are injurious to crops or human and animal health in their native area, or because they are natural enemies that were improperly or accidentally introduced, may become especially invasive and may greatly threaten biodiversity, health and economy3. The biomass is generally greater and the trade-off between growth and reproduction favours higher reproductive input in invasive plants compared to native plants3. For freshwater fishes, the tolerance to extreme temperatures and to changes in water quality, a large body size and a wide distribution in the native range have all been linked to establishment success and are considered robust indicators of it5. Similarly, invasion success by arthropods can be linked to reproductive strategies and resource acquisition. In their review paper, Comont et al.6 showed that ladybird species with broad diet range are likely to have a wider distribution than species with a more limited prey range6. For all of the above-mentioned taxa, in particular invertebrates, attributes of the invaded community, such as low community maturity and high niche opportunities, may play an important role in fostering invasion7. Additionally, the lack of regulation by natural enemies in the invaded area 8 and the consequent allocation of energy resources to fitness rather than defences 9 have been proposed to be the main determinants in the invasion success. Among insects, ladybird beetles may represent valuable model species to study different patterns of invasive processes10. Invading ladybirds invest high resources in reproduction and tend to show a large body size and high genetic diversity, phenotypic plasticity and likelihood to engage in competitive interactions with non-invading species12. Here, we focus on some aspects of the oviposition behaviour and reproductive physiology of ladybird beetles. The optimal oviposition theory predicts that females of predatory ladybird beetles would lay their eggs accordingly to habitat quality, prey abundance and suitability and the risk of competition with other predators, all of which affect offspring survival and development15. Although several studies have focused on comparing characteristics that make a patch \u201csuitable\u201d for a certain species, very few have focused on the ability of a female to acquire the information from the habitat and use it to locate a suitable oviposition site16. Theory predicts that ladybird species might exhibit differences in behaviour and ability to exploit a habitat for oviposition17. Species that respond more readily to an environment of good quality might confer to their offspring an advantage over juveniles of other competitors18. In fact, developing larvae might exploit preys that are free from competitors, allowing time to increase their body weight until the arrival of other predators19. This results in a competitive advantage, especially in the case of intraguild predation events due to an occasional shortage of the essential prey20.Some common traits, intrinsic to the species, are consistently related to the successful establishment of non-native organisms. Among plants, a recent meta-analysis underlined that invasive species have traits associated with higher performance over non-invasive speciesHarmonia axyridis (Coleoptera: Coccinellidae) is native to Asia and was widely introduced as a biological control agent of pest aphids, but it has spread also to many Countries where it was never intentionally released an intrinsic higher ability of H. axyridis against competitors28 and (2) a maternally driven decision in habitat choice for oviposition, which might indirectly confer a competitive advantage to offspring.The exotic predator H. axyridis, very little is known about its ability to exploit habitats for oviposition. We addressed this aspect here for this invasive species and for native Oenopia conglobata (L.) (Coleoptera: Coccinellidae). O. conglobata is potentially displaced by H. axyridis29 because of its low competitive ability in direct intraguild contests31 or field conditions32. Both ladybird beetles should be considered generalists, since their diet range largely extends to species other than aphids13. Both ladybirds also feed on psyllids and coccids, and O. conglobata is also frequently observed to prey on juvenile stages of leaf beetles, such as Plagiodera versicolor Laich. on willows. Additionally, both ladybird species have a preference for trees but may also occur on herbaceous plants34. In our experiments, we chose the system represented by Aphis fabae Scop. developing on Vicia faba L., as A. fabae represents, for both predators, a very suitable prey compared with other common aphids in terms of survival, larval growth, development and adult fresh weight36. We tested the prediction that H. axyridis, as more competitive than O. conglobata, makes better use of information from prey and from plants attacked by prey for a first assessment of patch quality. We performed a first group of experiments in a Y-tube olfactometer to test whether selected olfactory stimuli trigger the behavioural attraction of coccinellid females. The specific aim was to test whether odours emitted by the aphid, the plant and their interaction might represent kairomones or synomones, respectively, for the predators. Natural enemies are known to rely on a series of chemical cues used for long-range orientation to efficiently invest their limited time in the location of hosts and preys , after the removal of the infested leaf maintained their attractiveness toward O. conglobata than in H. axyridis , whereas no significant differences were observed after 48\u2009h .The best-fit model to explain ovarian development retained the principal effect of the coccinellid species, the plant treatment, the time post-set-up of the experiment, and the interaction between time and species Table\u00a0. The mod41 as well as predators42. For insect predators such as ladybird beetles, the choice of an optimal site to lay eggs is a crucial step that is important for the survival of their offspring17. In our study, H. axyridis showed a higher response than O. conglobata to olfactory cues associated with a plant-aphid system, both at the behavioural and physiological level.Volatile cues associated with the plant-aphid system have been shown to elicit a process of long-range host location for parasitoidsH. axyridis to aphid-infested plants suggests that at the aphid density chosen in our bioassay, this species is able to obtain information from volatile compounds. We observed that aphid and honeydew odours might act as a kairomonal message used by H. axyridis to locate prey. For this species, our results are consistent with those obtained by Leroy43, who identified a positive response to volatiles emitted from the aphid Megoura viciae Buckton, including Z, E-nepetalactone, [E]-\u03b2-farnesene, \u03b1-pinene and \u03b2-pinene, and 3-hydroxy-2-butanone, 3-methyl-butanal, 3-methyl-1-butanol and limonene emitted from aphid honeydew. In describing the mechanism of prey finding in H. axyridis, Obata45 demonstrated the involvement of visual and olfactory cues, in particular the attractiveness of odours from the infested plants and the odour from aphids combined with visual cues from a clean leaf. In our experiment, when the aphids and honeydew were removed from a V. faba plant, the plant maintained its attractiveness, suggesting the involvement of synomones emitted as a consequence of aphid feeding. Such synomones are systemically induced, as indicated by the attractiveness of H. axyridis to V. faba plants that were infested locally after removal of the infested leaf. The existence of induced volatiles emitted from plants that may be responsible for the attraction of H. axyridis females has only been documented with\u00a0(Z)-3-hexenol. This compound was produced in nettle plants that were mechanically damaged to tentatively simulate aphid infestation46, but probably (Z)-3-hexenol is not a reliable indicator of aphid infestation. In fact, an increase in its emission has been linked to damage from chewing herbivores also\u00a0(reviewed by ref. H. axyridis responds to non-specific cues because its broad diet range. A further explanation for H. axyridis attraction is that V. faba plants infested by A. fabae produce one or more specific compounds (herbivore-induced synomone) that might be reliable indicators of the aphid presence, but such compounds have to be identified.The strong response of O. conglobata showed significant attractiveness only to the treatment represented by aphids or by aphid-infested plants when tested vs. clean plants. Generally, this species showed a lower response to odours from aphids and/or plants compared to H. axyridis. A possible explanation might be that O. conglobata requires a complete set of odour cues from infested plants to assess a suitable habitat for oviposition. Aphids alone are not attractive for O. conglobata. Additionally, it appears that this species does not respond to induced synomones, thus explaining the lower ability in patch exploitation compared to H. axyridis. Olfactory sensilla used to perceive volatiles in the environment are probably located on the antenna. In H. axyridis females, those sensilla possibly include placodea, basiconica and coeloconica48. Antennal morphology combined with electrophysiology could reveal differences between O. conglobata and H. axyridis and possibly explain their different behaviour.By contrast, Harmonia axyridis is considered responsible for the displacement of native coccinellids in the United States49 and Europe50. Based on a recent risk assessment conducted on thirty native European ladybird species, the four species Adalia bipunctata L., Adalia decempunctata (L.), Calvia decemguttata (L.) and O. conglobata were identified as most at risk following the invasion of H. axyridis51. In Belgium, O. conglobata exhibits a negative trend in abundance in deciduous trees following the arrival of the invasive H. axyridis24. Predation on guild members by H. axyridis has been implicated as the primary cause for displacement of native species28, and the strength of these interactions has been recently described under open-field conditions54. Competition for resources has also been hypothesized10, and our results provide a partial explanation of this high ability for H. axyridis.H. axyridis exhibited higher plasticity in ovarian dynamics compared to O. conglobata. Intriguingly, here we also demonstrated that prey-related volatiles, which provide reliable cues for the presence of prey in the vicinity, are sufficient to induce physiological modifications in ladybirds. Oosorption in H. axyridis in the presence and absence of prey was devoted to allocation of nutrients for survival rather than for development, as has been previously described38. Here, we found that starved females of both H. axyridis and O. conglobata are able to modulate egg resorption based on the presence or absence of volatiles from aphids. The presence of prey odours reduced oosorption in both species, but when odours were absent, H. axyridis accelerated the resorption process compared to O. conglobata. Moreover, H. axyridis rapidly produced new eggs once food was provided again. Faster ovarian dynamics, which have also been observed in generalist vs. specialist ladybirds40, are useful because they enable species to quickly mature oocytes once they locate prey55.In tests of physiological responses, C. septempunctata responded more rapidly than the native Coccinella transversoguttata richardsoni Brown to changes in prey availability, by engaging in oosorption as a means of reserving resources under poor prey conditions39. This behaviour might have promoted the rapid and successful establishment of C. septempunctata in North America39. Similarly, the greater ability of H. axyridis to adjust the rate of oosorption compared to O. conglobata could contribute to the abundance and reproductive success of H. axyridis in natural and agricultural systems, which exhibit high variation in aphid densities. In this respect our results are in accordance with the greater flexibility hypothesis developed by Baker56.In North America, introduced populations of H. axyridis is more reactive than O. conglobata to olfactory cues from the plant-prey system, which might partially explain its higher competitiveness and consequent ability to dominate predator guilds in invaded habitats. Plants may indirectly affect the natural enemy community exploiting their herbivorous pests by mediating multitrophic interactions57. In this respect, the possibility of using plant volatiles to manipulate predator behaviour is promising when the purpose is to enhance the biological control of crop pests58 or control populations of invasive predators46. Therefore, the identification of prey-related volatiles responsible for the attraction of natural enemies would be necessary for a better understanding of prey location mechanisms by invasive vs. native species and for possible field applications.In conclusion, our results support our prediction that V. faba cv. Superaguadulce) were immersed for 24\u2009h in a slurry of water and soil (1:4) to favour germination and nodulation with Rhizobacteria. The seeds were individually planted in plastic pots (9\u2009\u00d7\u20099\u2009\u00d7\u200913\u2009cm) filled with a mixture of agriperlite, vermiculite, and sand (1:1:1) and grown in an environmental cabinet . Plants were watered daily and, from 1 week post germination, irrigated with an aqueous solution (1.4\u2009g/l) of fertilizer . For the experiments, three week-old broad bean plants with approximately six fully expanded leaves were used.Seeds of broad bean plants and reared under controlled conditions . Bugs were fed on an ad libitum diet of broad bean twigs infested with A. fabae. Food was changed every 2\u20133 days, and separate plastic cylindrical cages covered by a fine tissue mesh to allow ventilation were used for larvae and adults. After the emergence from pupae, male and female ladybirds were isolated for a few days with aphids and then paired in Petri dishes to allow mating. For all bioassays, groups of six physogastric females were detected and individually isolated in small vials 30\u2009min before bioassays and transferred to the bioassay room to be acclimatized. After behavioural observations, females were returned to Petri dishes with A. fabae to allow oviposition. Insects that did not oviposit within 24\u2009h were discarded from data analysis.Coccinellid cultures were established from Aphis fabae infested plant (IP), obtained by exposing all plant stems and leaves to 200 juvenile and adult aphids for 48\u2009h.Aphis fabae (APH) in a cage containing 200 juvenile and adult aphids. The cage consisted of a cylinder made of mesh positioned in the middle of a glass chamber.Aphis fabae honeydew (HON) obtained by positioning four plants as in treatment a) above a Parafilm foil for 48\u2009h. The Parafilm with the honeydew was positioned in the middle of a glass chamber.Clean plant (CP).Control (AIR), obtained by adding 200\u2009ml tap water inside the glass chamber to render the humidity of the control chamber similar to that of the treatments.In the second set of comparisons, the hypothesis that the plant itself might release synomones induced after the attack by aphids was tested. The treatments consisted of:Aphis fabae infested plant (IP), as in a).Aphis fabae and clean plant (CP\u2009+\u2009APH), obtained by positioning a cage with aphids as in b) close to a clean plant. The cage was attached to a wooden holder inserted into the soil. Care was taken to prevent the aphid honeydew from contacting the plant tissues.Injured plants (IP -APH), obtained as in a), but aphids were carefully removed 30\u2009min before the bioassay, both by washing the plant with tap water and using a fine paintbrush.Clean plant (CP), non-infested by aphids. If CP was associated with IP-APH, the plant was washed with tap water before the bioassay.In the third set of comparisons, we tested the duration of attractiveness of the plant attacked after aphid removal. The treatments consisted of:Injured plants (IP -APH), obtained as in h) and exposed respectively 24, 48, 72 or 96\u2009h after washing.Clean plant (CP), non-infested by aphids, washed and exposed respectively 24, 48, 72 or 96\u2009h after washing.H. axyridis responded positively to infested plants with the aphids carefully removed, the bioassays using j) and k) treatments were only conducted with this ladybird species.Given that only In the fourth set of comparisons, we tested whether the source of plant attractiveness is local or systemic. Treatments consisted of:th leaf to 50 mixed instars aphids inside a clip cage for 48\u2009h. Clip cage consisted of a 5-cm diameter x 1.0-cm height modified Petri dish, with the rim covered by a sponge ring and the bottom ventilated through a mesh-covered hole. The dish was supported by a hairpin attached to a wooden holder inserted into the soil. The clipped leaf was excised with forceps just before the bioassay.Locally infested plant (IP -CLIP), obtained by exposing the lower surface of the 4th leaf with a clip cage previously described in l). Similarly, the clipped leaf was excised with forceps just before the bioassay.Uninfested plant (CP -CLIP), obtained by clipping an untreated 4A first set of comparisons was conducted to test whether odours from aphids or plants are attractive to ladybird females. All odour sources were confined in glass chambers connected to the olfactometer device . The following treatments were performed:After the end of the treatment period, plants were transferred to the bioassay room.\u22121 using flowmeters. Plastic tubes were used to connect the device parts. The device was illuminated from above with two 22-W cool white fluorescent tubes and from below by an infrared source (homogeneous emission of wavelengths at 950\u2009nm provided by 108 LEDs). Before entering the olfactometer arms, each air stream passed through a cylindrical glass chamber with an O-ring sealed middle joint, containing different odour sources (see section \u201cTreatments\u201d). The stimuli were randomly assigned at the beginning of the bioassays and were reversed after testing three ladybird females. At every switch, the polycarbonate olfactometer was cleaned with water and detergent, and all glass parts were exchanged for clean parts. Glass parts were cleaned with acetone and heated overnight at 180\u2009\u00b0C. Ladybird females were singly introduced into the Y-tube olfactometer at the entrance of the stem and allowed to move freely for 5\u2009min. A monochrome CCD video camera fitted with a 12.5\u201375\u2009mm/F 1.8 zoom lens was used to record the insect behaviour. The camera lens was covered with an infrared pass filter to remove visible wavelengths. Analogue video signals from the camera were digitized by a video frame grabber59. Digitized data were processed by XBug, a video tracking and motion analysis system60. Insect response was measured in terms of residence time, i.e., the time spent by the female in each arm during the entire bioassay. The Y-tube olfactometer bioassays were carried out as paired choices, in which odour sources were always tested versus clean plants or air used as control.Female responses to volatile chemicals from different treatments were investigated using a Y-tube olfactometer made of a polycarbonate body sandwiched between two glass plates (height\u2009=\u20095\u2009mm) . Significance of the treatments higher than controls was evaluated through\u00a0planned orthogonal contrasts63.Bioassays were conducted daily from 09:00 to 17:00 under controlled conditions . Twenty-nine to forty-three replicates were conducted for each treatment. Females that did not move and those that did not exhibit any choice for at least one olfactometer arm during the first 3\u2009min of the bioassay, were discarded from the analysis. The percentage of time spent by a female in each arm of the olfactometer (Residence time) was calculated over the total experiment time\u00a0and logit transformed for the analysis. The number of active ladybird females, i.e. females that walked into one or both olfactometer arms, was scored, and\u00a0the percentage was calculated over the total number of replicates. Generalized Linear Models (GLMs) were fitted to test the effects of treatment vs. control arm within each source of volatiles per each ladybird species on the residence time (GLMs with Gaussian error distribution) and on the active females .\u00a0The \u201cinsect identity\u201d effect was included as random effect in generalized mixed effect models and its relevance was tested by means of likelihood ratio testV. faba plant. For each experimental setup, a total of 12 females, of both ladybird species, were individually positioned in a clean Petri dish (\u00d8\u2009=\u200990\u2009mm) with a bottom containing filter paper (Filter-Lab Filtros Anoia 1300/80) moistened with 2\u2009ml tap water were exposed to odour sources from either an infested or a clean H. axyridis and O. conglobata after a period of starvation. Females of both ladybird species ready to oviposit were isolated in Petri dishes and starved for 72\u2009hours to allow them to resorb all their eggs. Next, they were fed an excess of A. fabae and dissected 24 and 48\u2009hours later. The ovarian dynamic was investigated by observing the first oocyte per each ovariole, for a total of 16 ovarioles (i.e. 8 per ovary) randomly selected per insect. The different treatments were replicated 8 to 10 times.An additional experiment was performed to investigate the speed of new oogenesis in 64. All data were analysed using the R statistical environment65.For both experiments, the number of immature oocytes (undeveloped or resorptive) or mature oocytes divided by the total number of ovarioles was arcsine square root transformed before the analysis. For the first experiment, linear regression was performed to analyse the relationship between immature oocytes and the following variables: coccinellid species, plant treatment, hours from set-up (aphids removed) and the interactions between these variables. To identify the minimum adequate model, we used backward selection, starting with a model that included the coccinellid species, plant treatment, hours, and all interactions between the variables. For the second experiment, linear regression was performed to analyse the relationship between mature oocytes and the following variables: coccinellid species, hours from set-up (aphids added), and the interactions between the variables. The minimum adequate model was identified using backward selection. For both experiments, model selection was performed based on the Akaike Information Criterion (AIC)Supplementary Info"} +{"text": "Artificial Intelligence (AI) technologies and their related applications are now developing at a rapid pace. Indoor positioning will be one of the core technologies that enable AI applications because people spend 80% of their time indoors. Humans can locate themselves related to a visually well-defined object, e.g., a door, based on their visual observations. Can a smartphone camera do a similar job when it points to an object? In this paper, a visual positioning solution was developed based on a single image captured from a smartphone camera pointing to a well-defined object. The smartphone camera simulates the process of human eyes for the purpose of relatively locating themselves against a well-defined object. Extensive experiments were conducted with five types of smartphones on three different indoor settings, including a meeting room, a library, and a reading room. Experimental results shown that the average positioning accuracy of the solution based on five smartphone cameras is 30.6 cm, while that for the human-observed solution with 300 samples from 10 different people is 73.1 cm. With the application and development of technologies based on user location information, location-based services are now growing at a rapid pace. Especially in large and complex indoor environments such as museums, airports, shopping malls, and underground constructions, there is an urgent need for high accuracy location services. For outdoor environments where an open sky is visible, Global Satellite Navigation System (GNSS) can provide excellent positioning accuracy, however, GNSS signals are weak and can be easily blocked or attenuated by buildings . TherefoIndoor environments are characterized by all types of complex situations, such as obstacles, signal fluctuation or noise, environment setting changes, etc. . The comHumans can locate themselves in their ambient environment based on visual observations. In 1971, O\u2019Keefe found place cells that form a storage facility for location information. The human brain can constitute a complete map of an indoor environment, and activate a place cell when a location is identified. The indoor location information in the place cells is fused with the information of multiple nerve cells . May-BriSince a camera can obtain an image of an object, like the eye, can we also build an economical method that everyone can employ? To answer this question, firstly, various methods for optical indoor positioning systems have been investigated ,15,16,17Considering the popularization and development of the smartphone, we will choose it as the experimental equipment ,15. HoweThus, in this paper, inspired by the human brain, a visual positioning solution was developed based on a smartphone camera. The solution is based on the concept of locating objects with human visual observations, though a single camera cannot simulate the situation of two eyes. It collects visual observations (images) and processes the images with an algorithm developed by us, which is totally different from the processing process of the human brain. However, vision has its advantages ,19.Compared with the previous schemes, the following parts show the differences between the proposed systems. Firstly, the method based on the smartphone can achieve an accuracy that can satisfy human daily life. We chose the doorframe as a well-defined object instead of placing markers. While the location and orientation of the doorframe is available from the design map of the building, so that the local coordinate system that can be transformed to a global coordinate system. Thus, a user can locate themselves by taking a photo of the doorframe based on their smartphone camera. Finally, in order to investigate the potential of the smartphone camera for border perception via a test between the smartphones camera and the human visual observation to a well-defined object, the extensive experiments were conducted with five types of smartphones and 10 people in three different indoor settings. The average positioning accuracy of the smartphone camera solution is 30.6 cm, while that for the human-observed solution is 73.1 cm. The result is useful for future AI applications based on smartphones. This paper has five sections. The first section gives an introduction; the second section describes the smartphone camera solutions in detail; the third section explains the experiments; this is followed a discussion section; and, finally, the conclusion.It is assumed that there is a smartphone user in an indoor environment. He or she can take a picture of the doorframe with the smartphone. The size of the doorframe is available from the floor plan of the building. The pixel coordinates of the corresponding corners are obtained by the improved corner detection algorithm. Then the three angle elements and three direction elements of the smartphone can be acquired by the rational functions model (RFM). Finally, the user location in the doorframe coordinate system then can be obtained by the coordinate translation relationship.l and In this paper, the method for positioning mainly consists of the following four steps: Firstly, when the image is acquired, the door corners in pixel coordinates are determined. An improved corner detection of the image will be applied to extract the door corners. Secondly, the smartphone\u2019s exterior orientation elements are calculated, which include angle and linear elements. Finally, the relative position between the user and the door will be obtained, which is based on the transformation of the camera coordinate system to the object space coordinate system.Algorithm 1. Visual positioning algorithm.Acquire the side lengths of the door from the floor plan of the building and obtain the corner\u2019s pixel coordinates of the doorframe by the corner detection algorithm :Most smartphones on the market have a digital zoom that is able to enlarge the area of each pixel for image magnification. Since the lens of the camera is not perfect, the problem of image distortion occurs during the acquisition of the image . The disr is the radius of pixel.Therefore, in order to obtain accurate measurements in pixel coordinates, deriving the distortion parameters of the camera is required. The relationship between the pixel coordinates of the ideal image and that of the actual image is described in Equation (1), which considers two tangential distortions and three radial distortions:This work adopts the calibration method proposed by Zhang , which hTo further improve the accuracy of calibration, in particular, to reduce the calibration error caused by the problem of bending of the calibration plate itself and the coordinate error of the feature points, the method chooses an LCD to display the calibration template, which aims to maintain the high geometric precision and flatness of the template plane ,21. To obtain the pixel coordinates of the corners of the door, the method first uses the Harris corner detection method ,23, and In Harris corner detection, we calculate a round window Since By further assuming that M [Thus, the corner points can be detected according to the two eigenvalues of M . In thisFirstly, we compare the grayscale of individual pixel points and template nuclei in the template area to determine whether the pixel points belong to the USAN area, and the rules is:Secondly, we further calculate the number of pixels whose gray values are close to the center of the template:g = 16:Lastly, the point response function is used to eliminate the edges and internally redundant points. The threshold g is set to half of the number of pixels, i.e., After the corner detection, a round window with the radius equal to three pixels is used to calculate the average coordinate of corner point as the four door corners\u2019 pixel coordinates: R is the camera angle rotation matrix, T is the camera translation matrix, and can be obtained:x and y directions, and Equation (12) shows the transformation relationship between the pixel coordinate system and the camera coordinate system, where T. The basic principle of all the recovery algorithms for the rigorous imaging model are linearization of the collinear Equation (14). We adopt the classical rational polynomial model to restore the rigorous imaging model, i.e.:As shown in P is the weight of the observation. However, the control points have the same accuracy, thus P is the unit matrix. In this paper, considering the door is in the center of the picture, the starting value of T is a quarter of the total of the corner coordinates and the starting value of R is The least squares solution of the above parameters can be obtained:After obtaining the optimal solution of six exterior orientation elements, Equation (18) can be used to calculate the object space coordinates of the main point. Then we will acquire the relative position at the photograph moment between the smartphone and target:In this work, the method is tested in three typical office areas with different smartphones. As shown in We chose five different brands of smartphones in the field tests whose prices range from 1000 to 6000 CNY. As shown in It should be noted that although most smartphones are equipped with a digital zoom camera, in which the focal length is constant, different smartphones have different distortion parameters and different coverage areas. The black and white standard plate is projected in the center of the photograph when we make a calibration for the phone's camera lens. Therefore, during the field tests, the target should be projected in the center of the image area as far as possible to reduce the error of the distortion correction.This part mainly focuses on the evaluation of the relative position information acquisition ability and accuracy evaluation of smartphones in different experimental areas. In this part, we chose the iPhone 7P to experiment in three different environments. Each region is set with five lines whose angle with the door is 30\u00b0, 60\u00b0, 90\u00b0, 120\u00b0, and 150\u00b0, and there are six test points per straight line. Due to the size of each scene, there are different intervals between the testing points. In order to study the universality of the visual positioning method based on smartphones, here we use four other smartphones to test the method. We evaluated the method and tendency for error by the absolute value of the relative positioning accuracy in different areas and different smartphones.Meanwhile, by comparing the testing points of different lines, it can also be found that the relative position error becomes worse when the angle between the lines and the door decreases. As shown in In addition, There are many differences between the three scenes; in spite of this, smartphones show good performance in this test. All smartphone positioning accuracy can be below 50 cm in each scene. Thus, this method shows our smartphone can provide better positioning accuracy to us.In this paper, mainly in order to simulate the brain border cell function, an image sensor based on smartphones can maintain the smartphone in obtaining the relative position relationship of the border of the object, and provide a location information service for a human being. Thus, at each test point in scene 3, 10 testers were asked to estimate the relative position with the border by themselves. In In this section, we mainly highlight some of our experiences with the smartphone visual positioning. We will have a deeper discussion with respect to the experimental results.In this section, we discuss the error equation of the classical rigorous imaging model by the rational function model used in this paper. Additionally, we offer a discussion on the changing trend of the absolute distance error in distance and angle.The restoration of the rigorous imaging model by the rigorous imaging model is mainly a process of solving the accumulated error:H is the distance between the user and the door, and f is the focal length. Thus, the corresponding weighting matrix P is calculated as:At the beginning, we have calibrated the phone camera using the LCD screen. Thus, in this equation, we think that the correction of the principal point of the photograph coordinate and focal length are equal to 0. The number of control points is four x direction and error in the plane of y-O-z is as follows:Finally, the ratio between the errors in the r (cm/pixel) which is related to the relative distance with the door, the rational function model fitting error can be considered as the displacement of pixel points (pixel). The errors in the y-O-z plane and x, y, and z, which are caused by the fitting error of the rational function model are:y-O-z plane, and x, y, z direction. So, the positioning error of the smartphone is calculated as:If the sensor resolution is The formula above has three parts. Due to the baseline (S) being a constant value, the expression C shows that the C increases as the relative distance (H) between the smartphone and border increases. As the relative distance increases, the sensor resolution r will also increase. If we assume the pixel correction error , the testing points which have the same absolute distance from the door have the same parameter of C. However, because of the growing angle, the target in the picture will be smaller. Therefore, r will be larger with the greater angle between the door and testing point. Thus, the positioning error will become worse with the angle growth. Thus, the formula conclusion is also consistent with the conclusion of the comparison between In addition, the camera calibration scenario is in the region of about 80 cm between the phone and the target. However, the experiment distance ranges from 2 m to 13 m. The distortion area is changed by an automatic focusing function of the smartphone, the pixel replacement error becomes larger, which means that the relative positioning accuracy will be reduced to a certain extent.Despite the fact the various smartphones tested in various places have different relative position errors, the average accuracy is much higher than for humans, thus meeting the user demand. This method can not only acquire the relative position of the border, but also provide reliable border information for the indoor positioning based on the smartphone brain.In the above test, the difference in the accuracy in different scenes is mainly affected by the environmental factors. First, the quality of the target images will be affected by the surrounding environment. As shown in The difference in the various smartphones in the same place is mainly caused by the difference of the camera lens. First, the smartphones have different viewing angles. The iPhone 7P and XIAOMI 5 can obtain a picture containing the whole door in some places very close to the door, while the others cannot. Second, the difference of the lens includes different distortion parameters, while the distortion correction accuracy was slightly different. Additionally, the focusing algorithms of the five smartphones are different, which will lead to differences in the distortion correction.In this paper, the prediction of the relative location information using smartphones is generally better than that of the human brain. Although human beings are not very good at relating their relative position with the border, the brain fusion positioning system is still worth learning. With the improvement performance of the smartphone, the sensor of it becomes more abundant. Thus, the smartphone\u2019s perception of environmental information is bound to surpass human capabilities. Perhaps we can simulate our brain GPS system to make full use of the environment information that is perceived by the phone. In this paper, we simulated the function of border cells, and the result we obtained can be used in a smartphone indoor positioning system. In the future, we will simulate the system of the brain, and the result may be better.We have presented the visual location method based on a doorframe. This method achieved the function of border cells that obtain the relative position of the border. We experimented with multiple phones in different environments, and the result shows the universality of this method. On the other hand, by contrast with the border perception ability of the human brain, this method can be used to support the human indoor location perception service."} +{"text": "The lack of suitable and reliable scales to measure self-reported health and health behaviour among people with intellectual disabilities (ID) is an important methodological challenge in health research. This study, which was undertaken together with co-researchers with ID, explores possibilities for self-reported health scales by adjusting, testing, and reflecting on three self-reported health scales.In an inclusive process, the researchers and co-researchers with ID adjusted the SBQ (sedentary behaviour), SQUASH , and SRH scales, after which a test-retest study among adults with ID was performed. Test outcomes were analysed on suitability and test-retest reliability, and discussed with the co-researchers with ID to reflect on outcomes and to make further recommendations.N\u2009=\u200940) and test-retest reliability (N\u2009=\u200915) was higher for the adjusted SQUASH (SQUASH-ID), in which less precise time-based judgements are sought, than in the adjusted SBQ (SBQ-ID). Suitability and test-retest reliability were fair to moderate for the SRH-ID and CHS-ID. The main outcome from the reflection was the recommendation to use SQUASH-ID answer options, in which less precise time-based judgements were sought, in the SBQ-ID as well.Main adjustments made to the scales included: use easy words, short sentences, and easy answer formats. Suitability (This study served as a pilot of an inclusive process in which people with ID collaborated in adjusting, testing, and reflecting on self-reported health scales. Although the adjusted self-reported measurements may be reliable and suitable to the target group, the adjustments needed may impair measurement precision. This study\u2019s results contribute to informed decision making on the adaptation and use of self-reported health scales for people with ID. In the current patient-centred paradigm, self-reports such as patient-reported outcome measures and health behaviour are highly valued in care and research. Self-reports, often collected via questionnaires, can help to make shared decisions and tailor treatment plans , 2. SociIn data collection on health behaviour and patient-reported outcome measures for people with ID, questionnaires are often proxy-administered \u201311. It mSelf-reports of people with ID potentially contribute to the improvement of healthcare research for this group and the autonomy of people with ID, and answer their wish and democratic right to be involved in research \u201317. FurtImportant contextual factors for study designs in which people with ID participate are: access to the population, ethical concerns, and the abilities of the target group , 24. FirThis inclusive study on self-perceived health and health behaviours amongst people with ID consisted of three phases: (1) adjusting the three health scales; (2) performing an online test-retest study of the adjusted scales among people with ID; and (3) reflecting on the adjusted scales and the test-retest study results. To facilitate an inclusive approach, people with ID participated actively during the study as co-researchers in phaseThree health scales frequently used in the general population were selected by the researchers. Adjustments to the scales, the informed consent procedure, and the outline of the online questionnaire for people with ID were discussed by two co-researchers and the principal researcher, resulting in a list of recommendations according to which the researchers adjusted the questionnaire. Then, the adjusted questionnaire was pilot tested by three other co-researchers. Their feedback, together with recommendations from relevant literature , 23, 26,To test the adjusted scales among adults with mild ID, all adult participants in the Three Day March, part of the Dutch Special Olympics 2016, were invited to participate. The register for the Three Day March included email addresses for the participants\u2019 support person only. These support persons, often a family member or professional caregiver, received an invitation by email giving information on the study. They were asked to discuss participation in the study with the person(s) they supported and to discuss whether this person met the inclusion criteria of: having intellectual disabilities, being adult, being able to give informed consent, and being able to answers questions. When a support person served a group of up to five persons, a personalised invitation was sent for each person with ID. Support persons serving a group of more than five persons with ID received a general invitation followed by a phone call from the first author.Risks of, and objections to, participation were deemed to be negligible in our study, which asks respondents to fill out a questionnaire on health-related behaviour and self-reported health. Potential respondents with sufficient decision capacity according to their support persons were asked to give informed consent, as suggested by Iacono and Murray . After cThe original scales are the Sedentary Behaviour Questionnaire (SBQ), Short QUestionnaire to ASsess Health-enhancing physical activity (SQUASH), and a single-item scale on self-reported health (SRH). These scales are often used in health research \u201334. The \u2018On a typical weekday/weekend day, how much time do you spend doing the following?\u2019 Answer options are: none, 15\u00a0min or less, 30\u00a0min, 1\u00a0h, 2\u00a0h, 3\u00a0h, 4\u00a0h, 5\u00a0h, or 6\u00a0h or more. The item, total hours per week spent on sedentary activities, is calculated by multiplying weekday hours by five and the weekend day hours by two and summing these. Total hours spent on sedentary behaviour per day is calculated by dividing total hours per week by seven. Outcomes higher than 24\u00a0h per day are usually truncated to 24\u00a0h per day [The SBQ aims to measure the amount of time spent on nine sedentary activities: watching television, playing computer/video games, sitting while listening to music, sitting and talking on the phone, doing paperwork or office work, sitting and reading, playing a musical instrument, doing arts and crafts, and sitting and driving in a car, bus, or train. The question asked in the SBQ is: per day .commuting activities , (B) leisure-time activities , (C) household activities , and (D) activities at work and school . For each activity, questions are asked about the number of days per week (open answer box), average time per day (open answer box), and effort involved in the activity [The SQUASH assesses physical activity levels and may be used to measure compliance with physical activity guidelines . It contactivity .\u2018How would you rate your current general health on a scale from 1 to 10? \u2019 aims to measure self-reported health (SRH).Finally, the question The adjusted scales data, obtained in the online test and retest study, were analysed on suitability and reliability. Prior to analysis, data processing included the transformation of strings into numerical variables for the SBQ-ID according to the following rules: (1) answers such as \u2018no\u2019 and \u2018never\u2019 were given the numeric code \u20180\u2019; (2) for answers containing a range of values, the middle of that range was used, e.g. \u2018two\u2013three hours\u2019 yielded 2.5; and (3) soft quantifiers, such as \u2018rarely\u2019 and \u2018sometimes\u2019, were regarded as non-quantifiable answers. For the test-retest reliability of the SBQ-ID, missing values were coded as 0\u00a0h.\u03c4), between the two self-reported health scales (SRH-ID and CHS-ID). The statistical analysis was conducted using SPSS version 22.Indicators for suitability were response rate and the proportion of non-quantifiable and missing values, respectively. For interval measurements, the test-retest reliability was investigated by means of the Intraclass Correlation Coefficient (ICC) with a 95% confidence interval (CI). For categorical variables, the test-retest reliability was investigated by means of Kappa with a 95% CI calculated using bootstrapping . The ICCThe results of the test-retest study and the adjusted scales were discussed in two separate group discussions with two and three co-researchers respectively, the principal researcher and a moderator experienced in group discussions with people with ID. A PowerPoint presentation and A3 posters were used to show the participants the adjusted questionnaire and the results of the test-retest study. During the group discussions, the co-researchers reflected for each scale on the adjusted format and the results of the test-retest study and identified recommendations for further improvement. The transcription of the group discussions were thematically analysed on: (1) hours spent on certain activities, had to be adjusted as the time-based judgement sought might be too in-depth. Hence, the SQUASH question format was altered, whereas the SBQ format was maintained, allowing comparison of suitability of both formats.The discussion with the co-researchers with ID and the feedback from the pilot yielded the following general recommendations: (1) include questions to check whether the study information and the meaning of an informed consent is understood correctly, (2) group related questions, (3) depict per page or screen questions on one single theme only, and (4) explicitly allow the participant to ask for, and receive, help from a support person. Specific recommendations for the settings and layout of an online questionnaire were: (1) use of a clear font and large font size, (2) allow for item non-response, and (3) use multiple pages because scrolling down requires more motor skills than a single carriage return does. The co-researchers suggested many adjustments tapping clarity of language, such as use of easy words, easy answer formats, and short sentences. The co-researchers were indecisive on whether or not the SBQ and SQUASH, asking for days per week, average time per day, and effort to intensity and days per week. For each activity, respondents were asked to report on (1) the intensity with which they did this activity by choosing one of the tick box options: never, sometimes, often, or always; and (2) days per week, by ticking the days of the week when they normally do this activity (tick box with Monday\u2013Sunday).In the adjusted SQUASH (SQUASH-ID), the physical activities for which judgements were sought were the same as in the original SQUASH. However, the question format was altered from How many hours are you sitting on a weekday (Monday to Friday) when you are ...?\u2019) and an example was added. The co-researchers suggested changing the original multiple choice answer categories to an open answer box to allow respondents to express the time verbatim. Weekend days were split into Saturday and Sunday because activities on these days varied a lot according to the co-researchers.For the adjusted SBQ (SBQ-ID), the question phrasing was slightly changed \u2019 was rephrased as: \u2018What score do you give your own health? \u2019. As recommended by the co-researchers, one other question was added, namely, the health ladder, which has been used previously [How healthy do you feel?\u2019 with the instruction \u2018Place the arrow on the health ladder; green is very healthy, red is very unhealthy\u2019. The colours, or answer categories, on the ladder were green, yellow, light orange, dark orange, and red.The question eviously . The heaThese points were all taken into account in the programming of the questionnaire in Limesurvey . EstimatN\u2009=\u200916), the person with ID did not meet the inclusion criteria (N\u2009=\u200914), or the support person would be absent during the study period (N\u2009=\u20092). In total, 40 persons filled out the questionnaire of which 31 with help from someone else. The group consisted of 18 males and 22 females and their age ranged from 18 to 76 . Participants lived in a community group home (N\u2009=\u200915), independent with ambulatory support (N\u2009=\u200910) or with their parents (N\u2009=\u20097). For daytime activities most participants reported day-care (N\u2009=\u200919), and paid work (N\u2009=\u200913), where few reported voluntary work (N\u2009=\u20093) or school (N\u2009=\u20094). Out of the 40 respondents, 23 were willing to be approached 2 weeks later for the retest. Of these 23 persons, 15 persons answered the questionnaire twice.To pilot test the SBQ-ID, the SQUASH-ID, the Self-Reported Health scale for people with ID (SRH-ID), and the Coloured Health Scale for people with ID (CHS-ID), people with ID were invited to participate in this study see Fig.\u00a0. Some susoft quantifiers such as \u2018sometimes\u2019, \u2018not much\u2019; 2) time frames such as \u2018in the morning\u2019, \u2018before bedtime\u2019; 3) conditional answers such as \u2018depends on \u2026.\u2019, \u2018varies every day\u2019; 4) related to the respondents disability such as \u2018wheelchair bound\u2019, \u2018I cannot do that\u2019; or 5) associative answers such as \u2018coffee\u2019 when hours of sitting while eating and drinking was asked.For the SBQ-ID, missing values varied per question, from 2 to 12 out of 40 respondents. The provisions of non-quantifiable values also varied per question, ranging from 3 to 6 out of 40 respondents. These non-quantifiable answers were: 1) Eating or drinking (0.09) and Transport (\u2212\u20090.14) to substantial for Playing a musical instrument (0.79). Because of high numbers of non-quantifiable answers, the summary values hours of sedentary activity on a weekday/Saturday/Sunday/per week could be calculated for four to eight respondents: too few to calculate ICC.Due to missing values, the total hours of sedentary activities could be calculated only for 16 respondents and had a median of 10\u00a0h per day (IQR 6.00\u201315.61). One respondent reported a total time spent on sedentary activities per day that exceeded 24\u00a0h. The reliability test of the SBQ-ID showed heterogeneous results use the same answer type as used in the SQUASH-ID; 2) structure items in the categories \u2018commuting activities\u2019, \u2018activity at work, day-care and school\u2019, \u2018household activities\u2019 and \u2018leisure time activities\u2019; and 3) give examples of the items.In the SBQ-ID many missing and non-quantifiable answers had been reported. Looking at these results the co-researchers believed the question format, in which \u2018Do you walk in leisure time, that is not to get to school, work or day care?\u2019; and 3) providing example activities.Comparing the results of the SBQ-ID with the SQUASH-ID, the co-researchers valued the SQUASH-ID scale as much easier due to clearer answer options and requiring less detailed time-based judgements . Nonetheless, the co-researchers identified some possible difficulties in SQUASH-ID, including understanding what intense activities mean, understanding the difference between leisure time and work, and fitting in activities which are not specifically asked for in the items. Recommendations to improve the SQUASH-ID included: 1) clarify intense activities by listing physically intense activities; 2) change the questions on walking and biking in leisure time slightly, into Comparing the results of the CHS-ID and SRH-ID, the co-researchers considered the CHS-ID as easier than the SRH-ID. A suggestion to make the SRH-ID easier was to include a row of numbers or to combine the colour scale with the numbers. Differences between colours on the CHS-ID were unclear for one co-researcher which could be mitigated by the use of more contrasting green and red colours and by placing a line between the colours, or adding numbers. For the SRH-ID, the co-researchers reflected that respondents might not have given answers lower than 5 because a 6 is usually valued as sufficient and below 6 as insufficient and bad.The co-researchers provided possible explanations for the test-retest results. The co-researchers argued that people might have become aware of their own behaviour and therefore gave another answer the second time they answered the questions, which describes a research effect. Co-researchers also suggested changes in health state, leisure activities, or weather conditions, and, forgetting may have caused differences between test and retest answers.poor and almost perfect. In the reflection phase, building on the results of phase 2, further recommendations were done. Answer options that require less detailed memories and calculations, like days per week and intensity as used in the SQUASH-ID, seem to be more suitable to the cognitive abilities of people with mild ID than answer options in the SBQ-ID.This study aimed to explore possibilities for self-reported health scales by adjusting, testing, and reflecting on self-reported health scales in an inclusive manner. In the adjustment phase, the co-researchers with ID gave recommendations for the online questionnaire in general and specifically for the scales. Please note that the items of the SQUASH and SBQ were used as starting point. Pilot testing the adjusted scales on suitability among 40 persons with ID suggested that the SQUASH-ID was more suitable than the other scales, as non-response was higher in the SBQ-ID, the SRH-ID, and the CHS-ID. Pilot testing the adjusted scales on test-retest reliability among 15 persons with ID showed a test-retest reliability of the items of the three scales, varying between By using the described approach, we aimed to gain a better insight into what is needed to design measurement instruments that better fit the capacities of people with ID and how this may be achieved in an inclusive manner. The co-researchers provided a respondents\u2019 perspective by carefully and patiently discussing the scales, which, according to the literature, is a very important issue in adapting measurements for self-reports of people with ID , 26. In The results from this pilot study indicate that the better a scale is adjusted to the target population, the better the scale performs on suitability. In our study, the SQUASH-ID scale, in which less precise time-based judgements are sought, was more suitable than the SBQ-ID scale. Although caution should be taken when discussing the test-retest results because of the small sample size, it seems the SQUASH-ID scale performs better than the SBQ-ID scale. Although our results suggest that simplification of time-based judgements increases suitability and yields more reliable data, there is a cost also; it affects measurement equivalence to the original scales and reduces the precision of the concepts\u2019 measurement.N\u2009=\u200949 and 50, respectively) [In general, it is difficult to develop reliable items and scales to measure time-based judgements of behaviour , 43. Thectively) , 36. ThiTo the best of our knowledge, this study piloted an inclusive process in which people with ID contributed to the adjustment, testing, and reflecting on the suitability and reliability of self-reported health scales for people with ID. This study suffered from difficulties in recruitment, a commonly mentioned problem in studies among people with ID . DespiteThis study described a pilot of scale adjustment by means of an inclusive procedure. Further research is needed to test reliability and investigate validity of the SBQ-ID, the SQUASH-ID, the CHS-ID, and the SRH-ID in a large and diverse sample of people with ID. Testing responsivity of the scales in a longitudinal study is required to investigate whether these scales could be used in physical activity intervention studies. Although testing the scales in an online questionnaire may be convenient and time saving, testing the scales in a face-to-face mode should also be considered as this might improve response rate and decrease item non-response. In general, to increase the quality and availability of measurement instruments for this population, more projects are needed in which scales are adjusted together with people with ID and tested on reliability and validity.This study contributes to informed decision making on using self-reports and adjustments to self-reported health scales for people with ID. This pilot study\u2019s results indicate that commonly used self-reported measurements can be made suitable to people with ID in an inclusive process and may yield reliable scales. Nonetheless, scale adjustment may reduce measurement equivalence with original scales."} +{"text": "Endogenous cannabinoids (ECs) are lipid-signaling molecules that specifically bind to cannabinoid receptor types 1 and 2 (CB1R and CB2R) and are highly expressed in central and many peripheral tissues under pathological conditions. Activation of hepatic CB1R is associated with obesity, insulin resistance, and impaired metabolic function, owing to increased energy intake and storage, impaired glucose and lipid metabolism, and enhanced oxidative stress and inflammatory responses. Additionally, blocking peripheral CB1R improves insulin sensitivity and glucose metabolism and also reduces hepatic steatosis and body weight in obese mice. Thus, targeting EC receptors, especially CB1R, may provide a potential therapeutic strategy against obesity and insulin resistance. There are many CB1R antagonists, including inverse agonists and natural compounds that target CB1R and can reduce body weight, adiposity, and hepatic steatosis, and those that improve insulin sensitivity and reverse leptin resistance. Recently, the use of CB1R antagonists was suspended due to adverse central effects, and this caused a major setback in the development of CB1R antagonists. Recent studies, however, have focused on development of antagonists lacking adverse effects. In this review, we detail the important role of CB1R in hepatic insulin resistance and the possible underlying mechanisms, and the therapeutic potential of CB1R targeting is also discussed. Insulin resistance is a pathological condition characterized by the inability of insulin to regulate glucose and lipid metabolism in peripheral tissues even when insulin concentrations in the blood are elevated ,2. InsulEndogenous cannabinoids are lipid signaling molecules that regulate numerous biochemical processes, such as those involved in neuroprotection, pain, motor function, cardiovascular function, immune and inflammatory responses, energy balance, food intake, and cell proliferation ,5. The mThe activation of CB1R in the liver is associated with obesity and metabolic complications such as insulin resistance and dyslipidemia by promoting the fatty acid uptake, lipogenesis, and adipogenesis . Many stInsulin signal transduction is a complex mechanism regulated by numerous enzymes and modulatory proteins. The insulin receptor consists of two extracellular \u03b1 subunits and two transmembrane \u03b2 subunits, and binding of insulin to the receptor results in autophosphorylation on tyrosine residues and the subsequent tyrosine phosphorylation of insulin receptor substrates by the insulin receptor tyrosine kinase ,17. ReceSubsequently, the activated IRS-1 triggers signal transduction by binding to the catalytic subunit of PI3K, p110, and then phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3) . PIP3 is\u2212/\u2212) mice exhibit enhanced insulin sensitivity and tyrosine phosphorylation of IRS-1 in the liver and muscle tissues. Additionally, PTP1B\u2212/\u2212 mice are resistant to weight gain even on a high-fat diet. Moreover, PTP1B\u2212/\u2212 mice are hypersensitive to leptin, which is involved in the regulation of satiety and energy expenditure. Hence, PTP1B\u2212/\u2212 mice are lean and insulin-sensitive. The leptin signaling is also dependent on the tyrosine phosphorylation of IRS-1, which is mediated by the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway. Additionally, up-regulation of PTP1B has been reported in insulin resistance and obesity [Insulin resistance is a pathological condition characterized by the inability of insulin to elicit a hormone response in insulin-dependent cells to regulate glucose and lipid metabolism ,2. Insul obesity and, thu obesity ,25. PTP1 obesity .Previous studies suggest that several cytokines and inflammatory mediators in the plasma are up-regulated in insulin resistance, including tumor necrosis factor-\u03b1 (TNF-\u03b1), monocyte chemotactic protein-1 (MCP-1), C-reactive protein (CRP), and interleukin-6 (IL-6) . TNF-\u03b1 aOxidative stress can also contribute to insulin resistance by impairing insulin signal transduction . In thisIn addition to these factors, defects in serine phosphorylation of IRS-1 also plays a pivotal role in insulin resistance . Impairm\u2212/\u2212) exhibit a lean phenotype associated with reduced body weight and reduced fat mass [CB1R is found mainly in the central nervous system (CNS), particularly in several brain regions such as the olfactory bulb, hippocampus, basal ganglia, and cerebellum that show the highest CB1R expression . The cerfat mass . Additiofat mass . Moreovefat mass . In braifat mass . CB1R isfat mass .Apart from the CNS, CB1R is highly expressed in the peripheral nervous system and various peripheral tissues ,53,54,55Evidence suggests that ECs are directly involved in the physiological control of food intake and energy utilization by targeting central and peripheral sites, including skeletal muscle, the liver, and adipose tissue . CB1R acCB1R activation increases the activity of lipoprotein lipase (LPL), which promotes the hydrolysis of TGs into non-esterified fatty acids and their subsequent uptake by adipocytes, thus increasing fat storage in adipocytes ,73. CB1RInsulin resistance is a pathological condition, in which the cells do not respond to the insulin hormone ,2. Both It has been demonstrated that CREBH, the target transcription factor of CB1R, in conjunction with insulin resistance, also plays a pivotal role in the regulation of hepatic gluconeogenesis during fasting as well as diet-induced insulin resistance in rodent models . CB1R acThe triggering of hepatic CB1R signaling is also involved in the development of hepatic steatosis as well as insulin resistance and T2DM . HepaticCB1R is known to form a heterodimer complex with class A G protein-coupled receptor (GPCR) superfamily, which includes human orexin 1 (OX1) and OX2 receptors . Orexin Impaired insulin secretion by \u03b2-cells is a causal factor in the development of T2DM concomitant with insulin resistance. There are contradictory opinions regarding the synthesis of ECs in pancreatic islets, a process that varies from species to species. Few researchers claim that there is no synthesis of ECs in \u03b2-cells; however, CB1R expression has been shown in \u03b2-cells, of which activation enhances insulin release ,85. ReceAdditional evidence suggests that the CB1R activation in skeletal muscle decreases basal and insulin-mediated glucose uptake . CB1R knCB1R may be a promising therapeutic agent for obesity, insulin resistance, T2DM, and other metabolic syndromes. Rimonabant, an inverse agonist of central CB1R, has been recently approved as an antiobesity drug . SpecifiSchisandra chinensis, significantly improves hepatic CB1-mediated insulin resistance and gluconeogenesis [Natural products derived from medicinal herbs are considered less toxic and may cause fewer adverse effects. N-oleoyl glycine, a lipoamino acid, promotes 3T3-L1 adipogenesis and insulin-mediated AKT signaling that is associated with the activation of CB1R . More reogenesis . GN reveThe role of CB1R in the EC system has recently received a great deal of attention due to its role as a master regulator of whole-body and cell metabolism and the observation that it is associated with critical processes modulating food intake, energy expenditure, lipogenesis, glucose uptake, insulin resistance, and gluconeogenesis. Under pathological conditions, an enhanced expression of CB1R is observed in the hepatic cells, which contributes to the hepatic insulin resistance, fibrosis, lipogenesis, and steatosis. The evidence strongly indicates that pharmacological blockade of CB1R provides a promising approach against obesity and metabolic syndrome. It should be noted that CB1R-deficient mice are resistant to obesity and metabolic syndrome, and CREBH is a target of CB1R in the regulation of hepatic insulin resistance. ERR\u03b3 is also a downstream effector of CB1R and may play an important part in CB1R-mediated insulin resistance. The role of CREBH and ERR\u03b3 in insulin resistance and obesity deserves special attention. Recently, the interaction between CB1R and orexin receptor was reported. It was demonstrated that the inhibition of peripheral CB1R improves insulin sensitivity and glycemic control through the hepatic Sirt1/mTORC2/Akt signaling pathway. Moreover, CB1R can suppress insulin signaling by blocking the insulin receptor kinase activity. The CB1R antagonist, ibipinabant attenuates \u03b2-cell loss without affecting the body weight. Additionally, CB1R exerts its antiobesity activity by reversing the leptin resistance. Further, CB1R reduces the insulin sensitivity of skeletal muscles by targeting the PI3K-PKB axis and the Raf-MEK1/2-ERK1/2 pathway. Natural compounds such as GN and N-oleoyl glycine that exert an inhibitory effect on CB1R deserve further research and clinical trials, as they may possess therapeutic potential. In this review, we discussed the important role and the possible underlying mechanisms of CB1R in hepatic insulin resistance and metabolic syndrome that will provide clues for the treatment of CB1R-mediated hepatic insulin resistance without causing any adverse effect on the CNS."} +{"text": "NRF2 is a major transcription factor regulating the expression of antioxidative/detoxifying enzymes, involved in oncogenic processes and drug resistance. We aimed to identify molecular alterations associated with NRF2 activation in endometrial carcinoma (EC).POLE, TP53, NFE2L2, KEAP1 and CUL3 was performed using Ampliseq panels on Ion Torrent PGM (ThermoFisher). NRF2 activity was assessed by NQO1, GCLC, and AKR1C3 mRNA expressions, using TaqMan assays and quantitative RT-PCR.Ninety patients treated (2012\u20132017) for localized/locally advanced EC were included in this study. Formalin-fixed paraffin-embedded tissue samples were processed for immunohistochemical (NRF2 and Mismatch Repair proteins) analyses. Next generation sequencing (NGS) of a panel of genes including POLE exonuclease domain mutated , MMR-deficient (MSI-like) , TP53 mutated (Copy-number high-like) , and other tumors (Copy-number low-like) . NRF2 nuclear immunostaining did not correlate with NRF2 target genes expression. The 3 tumors with highest NRF2 target genes expression harbored oncogenic KEAP1 or NFE2L2 mutations. Low NQO1 mRNA and protein levels were observed in the TP53 mutated subgroup compared to others tumors (p < .05) and in silico analyses of The Cancer Genome Atlas data further indicated that NQO1 mRNA levels were lower in serous compared to endometrioid copy-number high EC.Tumors were classified as NFE2L2 or KEAP1 mutations. The subset of aggressive EC with low NQO1 mRNA level might represent a specific subgroup, which could be sensitive to combination therapies targeting oxidative stress.In contrast with previous reports based on immunohistochemistry, our study indicates that NRF2 activation is a rare event in EC, associated with POLE) catalytic subunit A mutations; 2/ a hypermutated group, characterized by a somatic microsatellite instability (MSI), largely due to methylations in MLH1 promoter; 3/ a group characterized by low copy-number alterations (Copy-number low group (CNL)); and 4/ a group characterized by high copy number alterations (Copy-number high group (CNH)) and TP53 mutations, that includes most serous carcinoma and some high grade endometrioid histologic subtypes, and that carries the worst prognosis. Despite these new insights, therapeutic breakthroughs are still awaited.Endometrial carcinoma is the most frequent gynecological cancer in woman. Two main histological types have been described, type 1 endometrioid carcinoma and type 2 including non-endometrioid subtypes with poorer prognosis so that positive values represent high expression and negative values represent low expression.qPCR were performed using TaqMan Gene expression assays (ThermoFisher Scientific) in a 20\u03bcL reaction with LightCycler 480 Probes Master on LightCycler 480 System Instrument (Roche Diagnostics), using 3 \u03bcL of cDNA, 1 \u03bcL of 20X TaqMan primer-probe mix, 10 \u03bcL of 2X LightCycler 480 Probes Master mix, and RNase-free water up to 20 \u03bcL. After incubation at 95\u00b0C for 10 minutes, the reactions underwent 45 cycles as follow: 10 sec at 95\u00b0C, 30 sec at 60\u00b0C, and 1 sec at 72\u00b0C,. Duplicate qPCR were performed on three core NRF2 target genes: p<0.05. All analyses were performed using R software v3.3.3.Correlations between qualitative and continuous variables were analyzed using Student t-test, or, if inapplicable, non-parametric test (Wilcoxon test). Correlations between continuous variables were analyzed using logistic regression. Correlations between qualitative variables were assessed using Fisher\u2019s exact test. Clustering analysis based on NRF2 target genes expressions used principal component analysis. Survival analyses were assessed by logistic regression using the Cox regression model. Primary event of interest was event free survival (EFS), defined as any event , censored by date of last news. Evaluation of the proportional hazard assumption was based on the Schoenfeld residuals method. Kaplan-Meier survival analysis used two-sided log-rank test. Statistical significance was defined by S1 Fig). Tumor classification was based on POLE exonuclease domain mutation, mismatch repair protein loss of expression and TP53 mutation, to retrieve TCGA molecular subgroups \u2013Fig 4F) when compared to patients without low NQO1 mRNA level = 4.66, 95%CI, p = 0.002)). However, in an adjusted Cox regression model that includes the TP53/CNH-like molecular group as main confounder, low NQO1 mRNA level was not independently associated with poorer survival , while TP53/CNH-like molecular subgroup was . These results show an association between low NQO1 mRNA level and poor outcomes but were underpowered to explore subgroup analysis according to NQO1 mRNA level within the TP53/CNH-like molecular group.Ninety patients were included in the survival analysis (EFS). Median follow-up was 24.1 months . As shoTP53/CNH-like tumors. This finding was also in contrast with conventional models suggesting that the high ROS production in aggressive tumor cells activates NRF2 in order to increase detoxification genes expression, such as NQO1, and to sustain a high proliferative rate [We report here a comprehensive analysis of the NRF2 activation process in endometrial carcinoma using genes expression data, genetic characterization, protein expression/subcellular location assay. In contrast with previous reports ,32, despive rate .TP53/CNH-like group, known to be of poor prognosis [gold-standard is for now available for NRF2 immunohistochemistry assay. In some reports, NRF2 staining has been observed in the cytoplasm. We considered the staining into the nuclei as consistent with NRF2 physiology, NRF2 being described as constantly degraded in the cytoplasm [in-silico data analysis, based on exome sequencing, while the qPCR expression results led to similar conclusions when faced to TCGA RNA-sequencing data. Our results indicate that assessing NRF2 core target genes expression appears more reliable than immunohistochemistry to assess NRF2 activation.We have observed a strong trend toward more NRF2 highly stained tumors within the rognosis . Howeverrognosis . No goldytoplasm or diffeet al. [et al. [TP53 missense mutations, NRF2 cooperates with TP53 mutant isoforms and colocalizes on proteasome gene promoters, subsequently inhibiting multiple tumor suppressive pathways and driving an aggressive phenotype. Importantly, this phenomenon precludes NRF2 binding on ARE promoters, and induces a downregulation of various AOS genes, more particularly phase II detoxification genes, such as NQO1 or HMOX1, or SLC7A11 [TP53/CNH-like tumors, especially tumors with serous histology, have very low NQO1 mRNA levels could be related to NRF2/TP53 cooperation and may have interesting clinical implications. The observation that serous carcinoma are initially highly sensitive to chemotherapy [in vivo by specific combination therapies [in vivo on triple-negative breast cancer models, a tumor carrying a molecular background similar to CNH endometrial carcinoma [NQO1 regulation may involve other transcriptional factors and its low expression in a subset of endometrial carcinomas might be related to biological processes independent of NRF2: an extensive chromatin and transcriptomic analysis of these tumors would be required to determine whether NRF2 transcriptional program is indeed skewed in TP53 mutated serous tumors. Finally, because the interplay described between NF-kB and NRF2 [TP53/CNH-like group could be affected by higher NF-kB activity, which could imply more inflammation. Further studies are warranted to confirm whether this feature could participate to aggressiveness of this molecular subgroup.Recent studies have reported the involvement of NRF2 in biological processes others than its canonical role in cell detoxification and metabolism reprogramming. As reported by Kalo et al. and more [et al. , in tumo SLC7A11 ,35. Subsotherapy could beherapies ,35. For arcinoma . Anotherand NRF2 , tumors NFE2L2/KEAP1 mutations leading to a strong NRF2 activation is a rare event in endometrial carcinoma, and that a subset of TP53/CNH-like endometrial carcinoma with serous histology have low NQO1 expression, that could be related to a NRF2/TP53 cooperation. This last phenomenon could drive their aggressiveness, although making them initially more sensitive to chemotherapy. If confirmed, new therapeutic approaches and combinations should be investigated in this subgroup of CNH endometrial carcinoma.In conclusion, our work suggests that S1 FigNGS: next generation sequencing, performed on IonTorrent PGM device. FFPE: formalin-fixed paraffin embedded. cDNA: coding DNA after reverse transcription. *one patient with 2 samples processed for sequencing, without qualitative changes on results. Sample with highest cellularity and best conservation was processed for RNA extraction.(PNG)Click here for additional data file.S2 FigNQO1, GCLC and AKRC3 expressions measured by quantitative RT-PCR are shown as 3D scatter plot. Axis: Color palette from dark blue to dark red follow NQO1 deltaCq expression. Plan is the NQO1 bidimensionnal regression plan according to GCLC and AKR1C3 expression .(PNG)Click here for additional data file.S3 Figcopy-number low-like tumors. MSI: microsatellite instable-like tumors. NRF2: tumors with NRF2 activating mutations , assumed to be clonal. NRF2_SC: tumors with NRF2 activating mutations , assumed to be sub-clonal on the basis of low allele ratio. POLE: POLE exonuclease domain mutated tumors. TP53: TP53/copy-number high-like tumors.Clustering was based on principal component analysis. PC1 and PC2: principal component 1 and 2. CNL: (PNG)Click here for additional data file.S4 FigKEAP1 (aa 324\u2013597) coding sequences or KEAP1 truncating mutations. Principal component analysis used RNA-seq RSEM (V2) data available at the www.cbioportal.org portal.Clustering was based on principal component analysis. PC1 and PC2: principal component 1 and 2. POLE: POLE molecular tumor group; MSI: microsatellite instable tumor; NRF2: tumors bearing a missense mutation within the NRF2/KEAP1 binding domains on NFE2L2 (DLG and ETGE motifs) or missense mutation on (PNG)Click here for additional data file.S5 Figwww.cbioportal.org portal. Genes considered belong to a NRF2 transcriptional signature including genes overlapping between two gene lists: genes repressed by a NFE2L2 siRNA-based silencing in A549 cells, a lung cancer cell line with a KEAP1 mutation :66\u201379) & genes significantly overexpressed in lung carcinoma KEAP1- or NFE2L2- mutated versus double wild-type carcinoma. Annotations depict mutations in signaling pathway of specific interest.Unsupervised hierarchical clustering depicted using heatmap. Data: RNA-seq RSEM (V2) gene expression data available at the (PNG)Click here for additional data file.S6 FigA-C: Cochin Hospital cohort (CCH). POLE: POLE exonuclease domain mutated tumors. MSI: microsatellite instable-like tumors. TP53: TP53/copy-number high-like tumors. CNL: copy number low-like tumors. Note that the 3 outliers in NQO1 and AKR1C3 plots are samples bearing clonal NRF2 pathway alterations . p-values are estimated using a post-hoc test adjusted for the risk \u03b1 for multiple comparisons. Note that POLE tumors were not included in the post-hoc test because of the number of samples in the group (<5). D-F: The Cancer Genome Atlas Cohort (TCGA). POLE: POLE tumors. MSI: microsatellite instable tumors. NS: p > 0.05 *: p \u2264 0.05. **: p \u2264 0.01. ***: p \u2264 0.001. ****: p \u2264 0.0001. ANOVA: analysis of variance. p-values depicted in * to **** for graphical purposes and estimated using post-hoc test adjusted for the risk \u03b1 for multiple comparisons.(PNG)Click here for additional data file.S7 FigType I and type II carcinoma: see (PNG)Click here for additional data file.S8 FigCCH: Cochin Hospital cohort.(PNG)Click here for additional data file.S9 Fig(PNG)Click here for additional data file.S1 Method(XLSX)Click here for additional data file.S2 Method(XLSX)Click here for additional data file.S3 Method(XLSX)Click here for additional data file.S1 DatasetClinical, histological and molecular characteristics of the 90 EC included in the study.(XLSX)Click here for additional data file."} +{"text": "Hepatitis E virus (HEV) is a zoonotic pathogen commonly considered an important foodborne virus. Pet dogs are important reservoirs of zoonotic agents. In the present study, the seroprevalence of HEV in pet dogs and pet veterinarians were found to be 28.2 and 5.0%, respectively. It remains unclear whether pet veterinarians are at higher risk of HEV transmission. However, pet animals and individuals who have contact with infected animals must be continually monitored for public health concerns. Hepevirus in the family Hepeviridae, which is a small non-enveloped, single-strand RNA virus [Hepatitis E virus (HEV) is commonly considered an important foodborne virus . From a NA virus . There aNA virus , 4. PartNA virus , 5.Antibodies against HEV were first identified in dogs in India in 2001 . SubsequSerum samples from dogs were randomly collected from small animal clinics in Seoul, Gyeonggi, and Jeonbuk provinces in South Korea. The sampling followed the General Animal Care Guideline as required and approved by the Institutional Animal Care and Use Committee of Chonbuk National University . A total of 287 serum samples were collected from pet dogs with or without gastroenteritis symptoms, such as diarrhea or vomiting, and were stored at \u2212\u200920\u2009\u00b0C until transportation to the laboratory. Pet veterinarians were asked to volunteer for this study, and serum samples were collected by convenience sampling from 40 participants working in companion animal clinics in Seoul, Gyeonggi, and Jeonbuk provinces in South Korea. This study was approved and performed according to the guidelines of the Institutional Review Board of Chonbuk National University (IRB no.: CBNU-2018-09-003-001). All sera were stored at \u2212\u200920\u2009\u00b0C until transportation to the laboratory.p\u2009<\u20090.05 were considered statistically significant.Anti-HEV IgG antibodies were detected using a commercial ELISA kit according to the manufacturer\u2019s instructions. Briefly, 100\u2009\u03bcl of dilution buffer was added into microplate wells, then 10\u2009\u03bcl of serum sample was added, gently mixed, and incubated at 37\u2009\u00b0C for 30\u2009min. After washing, 100\u2009\u03bcl of horseradish peroxidase conjugate was added and incubated for 30\u2009min and washed. One-hundred microliters of tetramethylbenzidine substrate was added and incubated at 37\u2009\u00b0C for 15\u2009min, then the reaction was stopped. Optical density (OD) was read at 450\u2009nm wavelength using a microplate reader (Model 680) , and the cutoff value was calculated as 0.16 plus mean OD value of the negative control based on maximizing true positives and minimizing false negatives. All sera, including a negative control serum, were tested in duplicate. Statistical analyses of seropositivity against HEV in dogs with or without gastroenteritis symptoms were performed using the Fisher\u2019s exact test. Values of p\u2009>\u20090.73, odds ratio: 1.16, 95% confidence interval: 0.60\u20132.24). Moreover, approximately 5% (2/40) of pet veterinarians tested positive for anti-HEV IgG\u00a0 and 27.6% (65/235), respectively. The seropositive rate of both groups was not significantly different (6/52 and Escherichia coli, and Staphylococcus aureus, between dogs and humans has been observed in some cases [Pet dogs are commonly considered a family member, and they have intimate contact with humans in most countries. Hence, zoonotic transmission of foodborne pathogens could be through direct contact with infected dogs or indirect contact with objects, such as contaminated food. In fact, the transmission of foodborne bacteria, such as me cases , 14. Morme cases . TherefoIn this study, all serum samples from pet dogs were collected from small animal clinics in the metropolitan area, and the overall seropositivity rate was 28%, which is similar to that observed in previous studies showing that seropositivity to HEV among pet dogs ranged from 19 to 30% in urbanized provinces in China . AlthougTo date, only two studies have investigated seroprevalence in pet veterinarians , 12. In Frequent exposure to animals infected with HEV can lead to an increased risk of viral transmission in humans according to several reports showing that the HEV seroprevalence among swine veterinarians were higher than those of the general population , 11, 12.In conclusion, HEV antibodies were detected in 28.2% of pet dogs in South Korea. While the sample size was relatively small, 5.0% of those tested were seropositive for pet veterinarians. The seropositivity rate among dogs with or without gastroenteritis symptoms was not significantly different. Although HEV seroprevalence in the general population was not examined in the present study, pet veterinarians are not at higher risk for HEV transmission. Therefore, our finding suggests that intensive monitoring of HEV infection and identification of HEV antigens in pet dogs is required to investigate the zoonotic potential of HEV transmission between humans and animals."} +{"text": "In addition, the carbon catabolite repression is alleviated by protease-based inverter-mediated flux redistribution under multiple carbon sources. By coordinating reaction rate using a protease-based oscillator in E. coli, we achieve d-xylonate productivity of 7.12\u2009g\u2009L\u22121\u2009h\u22121 with a titer of 199.44\u2009g\u2009L\u22121. These results highlight the applicability of programmable protein switches to metabolic engineering for valuable chemicals production.Synthetic biology aims to develop programmable tools to perform complex functions such as redistributing metabolic flux in industrial microorganisms. However, development of protein-level circuits is limited by availability of designable, orthogonal, and composable tools. Here, with the aid of engineered viral proteases and proteolytic signals, we build two sets of controllable protein units, which can be rationally configured to three tools. Using a protease-based dynamic regulation circuit to fine-tune metabolic flow, we achieve 12.63\u2009g\u2009L E. coli.Current flux rewiring technologies in metabolic engineering are mainly transcriptional regulation. Here, the authors build two sets of controllable protein units using engineered viral proteases and proteolytic signals, and utilize for increasing titers of shikimate and D-xylonate in To maximize product titer, yield, and productivity, metabolic flux needs to be finely reprogrammed without disrupting cellular homeostasis3. Current flux rewiring technologies have centered on the transcriptional-level regulation because of the ease and success of sophisticated engineering of metabolite-responsive transcriptional factors5. Despite unprecedented achievements making by transcriptional regulation7, the associated long response time can lead to faulted genetic circuits and ultimately fail to control metabolic flux8. Recently, multilayer regulation involving both transcriptional- and protein-level reprogramming is reported11. Protein-level regulation in those systems relies on the modification of target proteins with degradation tags or degrons14. However, these systems suffer from a number of issues, including tunability and orthogonality, and the lack of protein\u2013protein interaction regulators have limited the ability to design and engineer fast-response biomolecular switches that can rapidly reprogram metabolic flux and persistently maintain cellular homeostasis.Engineering microbial cell factory to produce valuable chemicals from renewable feedstocks plays an essential role to implement sustainabilityEscherichia coli. To unravel the design principles underlying protease-based metabolic switches, we have developed a synthetic biology toolbox, including protease-based dynamic regulation circuit (pbDRC), protease-based inverter (pbI), and protease-based oscillator (pbO), to achieve fast-response and tunable control of metabolic flux. These rationally designed switches exhibit superior applicability for the regulation of metabolic flux and improvement of metabolite production in the industrial workhorse E. coli.In this study, viral proteases that specifically recognize and cleave short cognate target sites are applied in combination with proteolytic signals to control protein stability and residence time in E. coli to accumulate or degrade target proteins when reprogramming metabolic flux, two basic protein regulatory units (including OFF-switch unit and ON-switch unit) were constructed. The OFF-switch unit consists of a protease that exposes the N-degron on the modified mCherry and drives its degradation expression variants with different strength of RBSs , which converts SHK to S3P in the metabolic pathway. Strains (DS1\u2013DS12) containing different combinations of SPPs and GPPs were assayed in NBS minimal medium without the addition of AAA and inducers, among which strain DS7 with a combination of promotor rpsT P2 and bolA exhibited a SHK titer increase from undetectable to 2.14\u2009g\u2009L\u22121 without cell growth sacrifice , an important starting material in treating influenza3P) Fig. . This evice Fig. , 14.Fig.rpsT P2), D14 , and DS7 (GPP-based repression of SHK kinase I combined with SPP-based induced degradation) were evaluated during the SHK production was achieved ; BL21 (derived from E. coli B); and HB101 (a hybrid of the E. coli K-12 and B) were engineered at the same sites, according to strain DS7. As shown in Fig. E. coli MG1655 by replacing the wild-type aroK with the rpsT P2 promoter-driven cleavable aroK. All strains with SPP-controlled TEVp exhibited a 3.6- to 8.5-fold titer (0.79\u20131.88\u2009g\u2009L\u22121) without acetate accumulation in comparison with the control strain in a d-xylose, d-xylonate, and glucose catabolic defects strain X2 could be achieved when induced after 6\u2009h of incubation (OD600\u2009=\u20090.78) , a 1.79-fold titer increase (4.25\u2009g\u2009L\u22121) was achieved over that of the control strain Fig. . On thisain Fig. . Those rd-xylonate titer could be achieved at an early stage of culture by co-expression of xylose dehydrogenase and lactonase20. However, given the fact that lactonase-mediated hydrolysis of d-xylonolactone to d-xylonate acidifies the cytoplasm, thereby decreasing cell viability, which ultimately compromises d-xylonate productivity21, the current d-xylonate titer and productivity produced by engineered E. coli was less commercially competitive by individual expression of xylose dehydrogenase (39.2\u2009g\u2009L\u22121 with 1.09\u2009g\u2009L\u22121 h\u22121)22 or simply by co-expressing xylose dehydrogenase and lactonase at a low level (108.2\u2009g\u2009L\u22121 with 1.8\u2009g\u2009L\u22121\u2009h\u22121)23. Therefore, increasing d-xylonate titer relied on the precise control of the cascade reaction rate.The third application is using pbO to tune carbon flux rate. Previous studies suggested that a higher d-xylonate and d-xylose catabolic defects was chosen as chassis for pbO demonstration (Table summv-(suF)tev-(teF)tvmv, the resulting strain, XO, harboring pbO was constructed , with a yield of 1.04\u2009g\u2009g\u22121 xylose, was achieved in the strain XO compared with that of the negative control strain XN , because they exhibited a biphasic relationship to pH from 3 to 8 . Moreover, by introducing protein oscillator, intracellular pH (in vivo pH) homeostasis was achieved in strain XO, which further led to the observed increase in cell viability by more than two orders of magnitude in 36\u2009h compared with that of the control strain XN medium at 37\u2009\u00b0C in shaker flasks , the input and output of protease-based circuits could be standardized to orthogonal proteases. Thus, by rationally configuring engineered viral proteases and proteolytic signals, distinct ON/OFF switches could be created and connected to each other to yield predictable behavior29. Recently, a series of protein circuits was constructed in mammalian cells by introducing leucine zipper motifs and split proteases30. Different from those studies, an alternative approach for constructing tunable protein circuits in E. coli was provided in this work by controlling the input protease dosage. Moreover, the types of protein circuits were further expanded by developing three protease-based biomolecular toolkits.The protease-based ON/OFF-switch exhibited good composability and extendibility in protein-level circuit construction. Compared with the transcriptional or posttranscriptional regulations reported in previous studies was genomically replaced by a glucose facilitator protein gene, Zmglf, from Z. mobilis. E. coli strains B001343, ATCC 873944, W311045, HB101, BL21, JM109, and DH5\u03b1 were used for the universal verification of pbDRC with the same operations. To test the genomic level regulation of pbDRC, the N terminus of aroK was tagged with a TEVp site (cleavage site: ENLYFQ) followed by the F-degron (FLFVQ). This modified aroK was genomically inserted into the place of wild-type aroK under the control of promoter rpsT P2 in chassis S4. Pathway enzymes, including aroGfbr, tktA, aroBopt, and aroE were selected for constructing enzyme overexpression plasmid pGABE. The backbone plasmid was a constitutive expression plasmid pJ01 (GenBank accession MK234843) with a pMB1 replication origin. The gene aroGfbr, a feedback-resistant mutant involving D146N, was obtained by rapid PCR site-directed mutagenesis46. The gene aroBopt, a codon-optimized variant, was obtained by optimization of the first eight codons (ATG GAG CGT ATT GTC GTT ACT CTG)47. An improved solubility and autolysis inactivated TEVp mutant 48 was codon-optimized and expressed under the control of SPPs on a low copy number plasmid, pTet-1 (GenBank accession MK234848). The N terminus of aroK was tagged with a TEVp site followed by an F-degron. This modified aroK was inserted into the pathway enzyme overexpression plasmid pGABE under the control of GPPs, including PrrnB P1, PrpsT P2, and PrpsJ.For SHK production, d-xylonate production, chromosomal genes including xylA (encoding xylose isomerase), xylB (encoding xylulose kinase), and ptsI (encoding EI in PTS system) was knocked out in E. coli BL21(DE3). All of the above gene deletion or replacement operations were carried out using CRISPR/Cas9 technology49. The xylB gene from C. crescentus was codon optimized, chemically synthesized, and cloned into the pTrcHisA vector to obtain pTrcHisA-ccxylB. The ptsI gene was cloned from genomic PCR amplification products. On the basis of this, the N terminus of xylB was tagged with a TVMVp site (cleavage site: ETVRFQ) followed by a F-degron. The N terminus of ptsI and tvmv were tagged with a TEVp site followed by a F-degron. The above three gene segments were assembled into a pTrcHisA vector using a one-step cloning kit. The xylC gene from C. crescentus was codon optimized, chemically synthesized, and N terminus modified by tagging with a TEVp site followed by a F-degron. SuMMVp (cleavage site: EEIHLQ) was applied for constructing protein oscillators. Successful gene cloning was verified by colony PCR, restriction mapping, and direct nucleotide sequencing.For \u22121 KH2PO4, 5.0\u2009g\u2009L\u22121 K2HPO4, 3.5\u2009g\u2009L\u22121 (NH4)2HPO4, 0.25\u2009g\u2009L\u22121 MgSO4 \u25aa 7H2O, 15\u2009mg\u2009L\u22121 CaCl2 \u25aa 2H2O, 0.5\u2009mg\u2009L\u22121 vitamin B1, 1\u2009mL\u2009L\u22121 trace element solution )50 containing 40\u2009g\u2009L\u22121 glucose and supplemented with 100\u2009mg\u2009mL\u22121 ampicillin and 30\u2009mg\u2009mL\u22121 chloramphenicol. All cultures were grown in 250\u2009mL shaker flasks at 200\u2009r.p.m. and at 33\u2009\u00b0C, which is the optimal temperature for SHK production.For SHK production in shaker flasks, seed cultures were grown overnight in LB medium at 37\u2009\u00b0C and then transferred into 50\u2009mL NBS minimal medium . Culture temperature was controlled at 33\u2009\u00b0C. Glucose (40\u2009g\u2009L\u22121) was added when the residual glucose fell below 10\u2009g\u2009L\u22121. Samples were taken as required and analyzed by high-performance liquid chromatography (HPLC).For SHK production in bioreactors, d-xylonate production in shaker flasks, seed cultures were grown in 50\u2009mL of LB medium in 250\u2009mL shaker flasks at 37\u2009\u00b0C on a rotary shaker at 200\u2009r.p.m. overnight. Then, 5% (vol/vol) of seed culture was inoculated into 50\u2009mL of NBS medium with 100\u2009mg\u2009mL\u22121 ampicillin and 30\u2009mg\u2009mL\u22121 chloramphenicol, and cultured at 37\u2009\u00b0C, 200\u2009r.p.m. When strain density reached 0.8, corresponding inducers were added. To optimize d-xylonate production, TB, 1.5\u2009\u00d7\u2009LB and NBS medium were used and culture conducted at either 37\u2009\u00b0C or 30\u2009\u00b0C.For d-xylonate production in bioreactors, seed cultures were prepared by incubating the strain XO in shaker flasks containing liquid LB medium overnight at 37\u2009\u00b0C. Then, 5% (vol vol\u22121) of seed culture was inoculated into 2\u2009L of the TB medium with 100\u2009mg\u2009mL\u22121 ampicillin in a 5\u2009L INFORS fermenter. The culture was first operated in a batch mode and the control settings were as follows: 37\u2009\u00b0C, stirring speed 600\u2009r.p.m., and airflow at 0.5\u2009v.v.m. During the culture process, the pH was controlled at 7.0 via automated addition of 30% NH4OH and 2\u2009M HCl. Antifoam 204 was added to prohibit foam development. When the dissolved oxygen started to increase at 4\u2009h (OD600\u2009=\u200914\u201315), 0.5\u2009mM IPTG, 200\u2009ng\u2009mL\u22121 anhydrotetracycline, and 70\u2009g\u2009L\u22121d-xylose were added for d-xylonate biosynthesis. Moreover, 500\u2009g\u2009L\u22121 glucose was fed into the fermenter at a constant speed of 8\u2009mL\u2009min\u22121 to achieve 2\u2009g\u2009L\u22121\u2009h\u22121 glucose supplementation until the end of culture. When the concentration of d-xylose fell below 20\u2009g\u2009L\u22121, 70\u2009g\u2009L\u22121d-xylose was added. Samples were taken to monitor cell density, residual sugar, and organic acid accumulation.For E. coli JM109 cells carrying corresponding plasmids were grown in 5\u2009mL LB plus antibiotics at 37\u2009\u00b0C for 12\u2009h in an Innova 44 shaker at 200\u2009r.p.m. The culture was diluted 1:100 in 200\u2009\u03bcL of fresh LB plus antibiotics and grown at 30\u2009\u00b0C for 3\u2009h with vigorous shaking at 1000\u2009r.p.m. in a Titramax 1000 incubator . For time-course measurements, this was the t\u2009=\u20090\u2009h time point. To maintain the cells in exponential growth phase, the culture was diluted 1:5-fold every 2\u2009h. Samples were taken as required and were further diluted 1:10 in 200\u2009\u03bcL of fresh LB with antibiotics and inducers for measurement using a SpectraMax M3 microplate reader . GFP abundance was quantified at an excitation wavelength of 488\u2009\u00b1\u200910\u2009nm and an emission wavelength of 511\u2009\u00b1\u200910\u2009nm. YFP abundance was quantified at an excitation wavelength of 515\u2009\u00b1\u200910\u2009nm and an emission wavelength of 544\u2009\u00b1\u200910\u2009nm. mCherry abundance was quantified at an excitation wavelength of 588\u2009\u00b1\u200910\u2009nm and an emission wavelength of 644\u2009\u00b1\u200910\u2009nm.600 reached 0.6, 0.5\u2009mM IPTG and 200\u2009ng\u2009mL\u22121 ATC were added. For time-course determinations, this was the t\u2009=\u20090\u2009h time point. Continuous sampling was performed for fluorescence detection on a SpectraMax M3 microplate reader.For oscillation analysis, strains were cultured in LB medium in 250\u2009mL shaker flasks at 30\u2009\u00b0C, 200\u2009r.p.m. When OD600 of 0.2. The assays were performed using a LSR Fortessa instrument (BD Biosciences) using fluorescein isothiocyanate (FITC) (GFP) and PE-TxRed (mCherry) channels. The voltage gains for each detector were set to FITC, 407\u2009V and PE-TxRed, 650\u2009V. Compensation was performed using cells that express only GFP or mCherry. For each sample, at least 10,000 counts were recorded using a 0.5\u2009mL\u2009s\u22121 flow rate. All data were exported in FCS3 format and processed using FlowJo software .For flow cytometry analysis, sample cells were washed twice with phosphate-buffered saline (PBS) and resuspended to an ODThe molten LB medium was poured onto a glass slide after adding appropriate antibiotics (Amp and Cm) and inducers (IPTG and ATC). This agarose pad was solidified at room temperature. When the density of exponentially growing strains reached 0.8, inducers of IPTG and ATC were added. Then, 1\u2009\u03bcL of cultures were pipetted onto an agarose pad and a cover slide was put on the top softly to prevent evaporation. Strain was in situ cultured at 30\u2009\u00b0C and this time was set as zero-time point. Microscopy images were taken using a Nikon ECLIPSE 80i microscope equipped with a \u00d7100 oil-immersion objective. Phase-contrast and fluorescence time-lapse images were recorded every 30\u2009min using a Nikon DS-Ri1 camera. Bright-field images and GFP fluorescence images were analyzed using ImageJ software.600 reaching 0.3 (2\u2009h), 1\u2009mL of culture was sampled, centrifuged at 5000\u2009\u00d7\u2009g for 5\u2009min, and resuspended in PBS . Samples were centrifuged again under the same conditions and then inoculated into a fresh shaker flask with LB media with no IPTG but 200\u2009ng\u2009mL\u22121 ATC.For testing the effects of inducing TEVp on the degradation of fluorescent proteins, cultures were first grown in the pre-induction condition in shaker flasks , where target mCherry protein was expressed. Upon the OD\u22121 ATC and 0.5\u2009mM IPTG). The fluorescence and OD600 at the zero-time point was determined by extrapolating a negative control condition (no TEVp expression) back to t\u2009=\u20090\u2009h.For testing the effects of inducing TEVp on the accumulation of fluorescent proteins, cultures were first grown in LB media in shaker flasks overnight. Then, cells were inoculated into 250\u2009mL shaker flasks (working volume of 25\u2009mL) at a dilution of 1:100 in LB media . Crude extracts were lysed by sonication and centrifuged at 12,000\u2009\u00d7\u2009g for 10\u2009min. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard. SHKkinase activity51 was measured in a 1\u2009mL reaction mixture containing 4\u2009\u03bcM ATP, 1\u2009\u03bcM SHK, 10\u2009\u03bcM NaF, 5\u2009\u03bcM MgCl2, 25\u2009\u03bcM barbital buffer (pH 9.0), and cell extract with 0.1\u20131.0\u2009mg of protein. One unit of SHK kinase corresponded to 1\u2009\u03bcM of SHK consumed per minute.Strains were cultured in NBS medium and collected every 6 or 12\u2009h, centrifuged at 12,000\u2009\u00d7\u2009\u22121 propidium iodide was added to diluted samples and incubated for 15\u2009min. Images were acquired using a Nikon ECLIPSE 80i microscope equipped with a Nikon DS-Ri1 camera. The viability of cells was assessed by inoculating LB plates with cells grown in liquid NBS. Cells were diluted to OD600\u2009=\u20090.5 and 10\u2009\u03bcL of 10\u00d7 serially diluted cell suspension was spread on each agar plate. Plates were incubated at 37\u2009\u00b0C for 12\u2009h before counting.For vitality staining, 100\u2009ng\u2009mLR410/470 values) of pHluorin fluorescence emitted (510\u2009nm) under excitation at 410 and 470\u2009nm was used to measure intracellular pH.Cells were incubated on ice for 5\u2009min, centrifuged, and resuspended with cold PBS buffer. The collected cells were incubated at 37\u2009\u00b0C for 30\u2009min with solutions containing 150\u2009mM KCl, 20\u2009\u03bcM nigericin, and 50\u2009mM buffering agents . The ratio , equipped with an Aminex HPX-87H ion-exchange column and a refractive index detector. Analysis was performed with a mobile phase of 5\u2009mM sulfuric acid (65\u2009\u00b0C) at a flow rate of 0.6\u2009mL\u2009min\u22121 and detected by monitoring absorbance at 210\u2009nm.The concentrations of SHK, acetate, glucose, Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Source Data"} +{"text": "We assessed the diagnostic performance of each MRI variable and the different combinations. Our results showed that the relative cerebral blood volume (rCBV) in the true progression group was significantly higher than that of the pseudoprogression group (0.541\u2009\u00b1\u20090.154) (p < 0.001). Among the 18 patients who had serial DSC-MRI, the rCBV of the progression group differed significantly from pseudoprogression group (p=0.015). With an rCBV threshold of 0.743, the sensitivity and specificity for discriminating true progression from pseudoprogression were 76.5% and 92.9%, respectively. The Cho/Cr and Cho/NAA ratios of the true progression group were higher than those of the pseudoprogression group ((p=0.001), (p < 0.001), respectively). The areas under ROC curve (AUCs) of enhancement pattern, MRS, and DSC-MRI for the differentiation were 0.782, 0.881, and 0.912, respectively. Interestingly, when combined enhancement pattern, MRS, and DSC-MRI variables, the AUC was 0.965 and achieved sensitivity 90.2% and specificity 100.0%. Our results suggest that DSC-MRI can significantly improve the diagnostic performance for identifying glioma progression. DSC-MRI combined with conventional MRI may promptly distinguish true gliomas progression from pseudoprogression when the suspected gadolinium-enhancing lesion was found, without the need for a long-term follow-up.Accurately and quickly differentiating true progression from pseudoprogression in glioma patients is still a challenge. This study aims to explore if dynamic susceptibility contrast- (DSC-) MRI can improve the evaluation of glioma progression. We enrolled 65 glioma patients with suspected gadolinium-enhancing lesion. Longitudinal MRI follow-up or re-operation ( Gliomas are the most common primary brain tumors in adults . At presAs a golden standard, histopathology is limited in clinical practice for the disadvantage of invasive sampling, sampling error, and high cost . MRI is Previous studies have shown that MR perfusion-weighted imaging, especially dynamic susceptibility contrast perfusion MRI (DSC-MRI), can reflect tumor angiogenesis which is valuable for glioma grading, estimation of prognosis, and differentiating tumor recurrence from treatment-related changes , 9, 10. This study was approved by the Institutional Review Board. The informed consent was waived for its retrospective nature. A total of consecutive 65 glioma patients with suspicious gadolinium-enhancing lesion were recruited , according to the following eligibility criteria: (1) gliomas were confirmed by histology; (2) gross-total resection of tumors ; (3) com1WI, T2WI, FLAIR, DWI, MRS, and DSC-MRI. The parameters of DSC-MRI: perfusion-weighted gradient-echo echo-planar sequence; repetition time, 2000\u2009ms; echo time, 40\u2009ms; slice thickness, 6.5\u2009mm; the data was acquired every one second for a total of 1\u2009min 30\u2009sec, with Gd-DTPA injected with a MRI-compatible power injector at a rate of 3\u2009ml/sec, followed by a 20-ml 0.9% saline flush using same flow rate.All studies were performed on a 3T MRI scanner , using an 8-channel phased-array coil for acquisition. The conventional MR protocol included precontrast and postcontrast T1WI, T2WI, and FLAIR. The multi-parametric MR imaging protocol included TT2\u2217-weighted sequence. We placed 3 circular ROIs in the region with the largest enhancement . The enhancement patterns of residual cavity wall were divided into thin-linear , thick-linear , and nodular wall enhancement (with nodular enhancement of 5\u201310\u2009mm in thickness) patterns . Cerebraancement , and thet-test when they were in non-normal distribution, or using Mann\u2013Whitney U test when they were in non-normal distribution. The interobserver consistency between the two neuroradiologists was evaluated with intraclass correlation coefficient (ICC). The survival times were estimated with the Kaplan\u2013Meier methods. Receiver operating characteristic (ROC) curve analysis was employed in determining the best cutoff values of rCBV and metabolite ratios in differentiating true progression and pseudoprogression by maximizing the sum of sensitivity and specificity. The diagnostic performance of all variables was measured as area under ROC curve (AUC). The level of significance was set at p < 0.05.Statistical analysis utilized the software SPSS for windows release 25.0 . Categorical variables were analyzed with log-rank test. The quantitative data between true progression and pseudoprogression groups, including rCBV, Cho/Cr, Cho/NAA, and ADC, were compared by using two-tailed Student's p=0.403), radiation dose (p=0.615), histopathologic grade (p=0.451), and Karnofsky performance status (KPS) scores (p=0.154).The median follow-up span was 590 days (range: 210\u20132670\u2009days). The median progression-free survival (PFS) was 360 days [95% confidence interval (CI): 399\u2013580 days] and median overall survival (OS) was 590 days (95% CI: 603\u2013790 days) . 22 patip=0.067) in spite of lower ADC value for true progression group (p \u2264 0.002) than those of pseudoprogression group (p=0.485). Using a cutoff Cho/Cr value of 2.475, a sensitivity 51.0% and specificity 92.9% were achieved; using a cutoff Cho/NAA value of 2.155, a sensitivity 64.7% and specificity 100.0% were achieved for separating the two groups.Among the patients with DWI data (54/65), there was no significant difference in ADC between two groups (on group . The Choctively) , whereasp < 0.001), so did between the normal brain tissue and pseudoprogression lesions (p < 0.001). The rCBV of true progression group was significantly higher than that of pseudoprogression group (0.541\u2009\u00b1\u20090.154) (p < 0.001) (p=0.015) between true progression and pseudoprogression groups. The between-group comparison revealed a significant difference between the pseudoprogression and the true progression groups corresponding to a large effect size (Cohen's d\u2009=\u20091.392). The ROC analysis showed that, using a cutoff rCBV value of 0.045, it achieved AUC 0.904, sensitivity 100.0%, and specificity 75.0% to separate the two groups.The rCBV of normal brain tissue was 0.993\u2009\u00b1\u20090.106 (0.854\u20131.209). There was significant difference between the rCBV of normal brain tissue and that of true progression lesions (< 0.001) . Among 6The agreement was excellent between the two neuroradiologists for evaluation of the functional MR variables, including rCBV , ADC , Cho/Cr , and Cho/NAA .Figures 1W imaging, and MRS can greatly improve the diagnostic performance in distinguishing true progression from pseudoprogression.We applied DSC-MRI, along with conventional MRI sequences, DWI, and MRS, to assess the impact of perfusion parameter on the differential diagnosis of true progression versus pseudoprogression in patients with gliomas. Our results suggested that a combination of DSC-MRI, contrast enhanced TAccurately and quickly distinguishing true progression from pseudoprogression in glioma patients after standard chemoradiotherapy remains a major clinical challenge . Newly eThe DSC-MRI has been widely used in evaluation of the suspicious lesions in posttreatment glioma patients , 18\u201320. Longitudinal change in rCBV may improve the discrimination between true progression and pseudoprogression lesions , 22. TheMRS metabolites ratios provide useful information in distinguishing true progression from pseudoprogression lesions , 23, 24.The role of DWI in differentiating true progression from pseudoprogression lesions remains ambiguous. Most studies showed the accuracy of ADC value in the differentiation was the lowest among all functional MR techniques , 8, 23. Since conventional morphologic imaging findings are often limited in differentiation of true progression from pseudoprogression lesions in gliomas, various advanced MR imaging modalities, including DWI, MRS, and DSC-MRI, can provide additional physiologic or metabolic information about posttreatment gliomas. However, the diagnostic accuracy of each technique is still limited and different , 15, 28.Several limitations of this study should be mentioned. First, the number of the patients in this retrospective study was relatively small because we only recruited those patients with DSC-MRI and also with sufficient MRI follow-up data. Second, the final diagnosis of most patients (95.4%) in the current study was based on the follow-up data. Third, we measured CBV and DWI in a 2D mode, which could not reflect local pathologic changes completely. Fourth, although the longitudinal change in rCBV could improve the diagnostic performance, only 18 of 65 patients in our cohort had the sequential DSC-MRI data.In conclusion, DSC-MRI, especially longitudinal change in rCBV, showed satisfactory diagnostic performance and can be a useful tool in distinguishing true glioma progression from pseudoprogression. Moreover, the combination of DSC-MRI and conventional morphological imaging features and other advanced MR techniques would further improve identifying the progression, which may greatly facilitate the individualized management of posttreatment glioma patients. This combination may even eliminate a long-term follow-up when a suspected gadolinium-enhancing lesion is found."} +{"text": "Amino-coupling conjugation was carried out between amino-modified oligonucleotides (CatG4-NH2) and carboxylated quantum dots (CdTe@COOH QDs). The obtained products were characterized by spectroscopic methods , and IR) and the transmission electron microscopy (TEM) technique. A QD-DNA system with a low polydispersity and high stability in aqueous solutions was successfully obtained. The catalytic activity of the QD-DNA conjugate was examined with Amplex Red and ABTS ) indicators using reactive oxygen species (ROS) generated by visible light irradiation. The synthesized QD-DNAzyme exhibited enhanced catalytic activity compared with the reference system (a mixture of QDs and DNAzyme). This proved the assumption that the covalent attachment of DNAzyme to the surface of QD resulted in a beneficial effect on its catalytic activity. The results proved that the QD-DNAzyme system can be used for generation of the signal by light irradiation. The light-induced oxidase activity of the conjugate was demonstrated, proving that the QD-DNAzyme system can be useful for the development of new cellular bioassays, e.g., for the determination of oxygen radical scavengers.Here, we report the synthesis of a quantum dot (QD)-DNA covalent conjugate to be used as an H The group of ROS mainly includes superoxide anion radicals ,5,6. Thee (H2O2) ,8,9, chee (H2O2) ,11, and e (H2O2) ,13 approe (H2O2) ,15.2 NP [Nowadays, semiconductor nanoparticles (NPs), for example, ZnO NP ,17,18, T2 NP ,20,21, a2 NP ,23,24, a2 NP ,23,24. T2 NP ,26,27. A2 NP . Conside2 NP . They ex2 NP used simIt is unclear whether the switching enzymatic activity of HRP/QDs by light is a unique phenomenon of protein enzymes ,29, or wIn this study, we examined the peroxidase activity of G-rich oligonucleotides attached to QDs. This system was designed as an oxidase-mimicking DNAzyme activated by light due to the QD generation of ROS . The DNAIt has been proven that CdTe nanoparticles with a diameter of 2.7\u20133.0 nm possess a band gap of 2.4 eV and, upon visible light irradiation, are able to generate electrons which interact with oxygen and water molecules to form superoxide, hydroxyl radicals, singlet oxygen, and ROS . It is aIn order to optimize the conditions of QD-DNA synthesis, the molar ratio of DNA:QD was investigated. The main goal was to obtain the conjugate product with the lowest polydispersity. A series of syntheses with different amounts of DNA equivalents were performed and analyzed using agarose gel electrophoresis . The mobIn order to verify whether oligonucleotide molecules were covalently attached to the QD surface, the obtained QD-DNA conjugate was examined by a separation experiment with magnetic particles . DetailsA260 and A350 are the absorbance for the QD-DNA conjugate, and \u03b5 parameters denote molar extinction coefficients for quantum dots (QD superscript) and CatG4 (G4 superscript) at 260 or 350 nm (subscripts). The values of extinction coefficients were determined from calibration graphs , dynamic light scattering (DLS), and zeta potential measurement . The zetThe spectral properties of the QD-DNA conjugate and related systems were studied using UV-Vis absorption spectrophotometry, the fluorescence, and circular dichroizm (CD) spectroscopy. The UV-Vis spectrum of QDs displayed broad absorption, extending from the ultraviolet down to the band edge around 350 nm and a low intensity absorption band at 485 nm A, an addAs can be seen in SV = 6.0 \u00d7 104 M\u22121, G-quadruplex: KSV = 4.5 \u00d7 104 M\u22121, and duplex: KSV = 8.9 \u00d7 104 M\u22121). The double-stranded form of DNA is able to quench fluorescence almost two times more efficiently than single-stranded oligonucleotides, but one should consider that dsDNA possesses two times more nucleobases than G4 and ssDNA forms of CatG4. Therefore, we assumed that this quenching was caused by the physical adsorption of DNA nucleobases on the surface of QDs, followed by a photoinduced electron transfer between QDs and nucleobases. Both forms of G-quadruplex and random coiled oligonucleotides quenched QD fluorescence, with a subtle difference showing that the G-quadruplex structure quenched the fluorescence less effectively. This difference also confirms that the process of the attachment of oligonucleotides to QDs was carried out successfully. The Zeng research group [Fluorescence quenching titration of QDs was performed for three structural forms of CatG4: G-quadruplex (in the presence of K+); random coil conformation (without stabilizing ions); and with dsDNA (CatG4/cCatG4 hybrid). The high values of the Stern\u2013Volmer constants suggested static quenching . All thrch group noticed SV value obtained for G4 DNA quenching . Nemeyer et al. [+\u25cf to the colorless ABTS2+ product. DNAzymes usually display lower activity than HRP. Therefore, we could not observe such an initial absorbance increase. Another reason for the lower activity of QD-DNAzyme compared with that for the QD/HRP system stems from the different method of QD irradiation, as well as the different type of QDs, used by Nemeyer et al. (CdS produces a higher number of ROS than CdTe QDs) [All peroxidase activity experiments were carried out for the systems that contained hemin at a 1:1 ratio to G4 DNA. The association of hemin changed the QD-DNA conjugate into a QD-DNAzyme catalyst. Two substrates for the catalytic reaction were studied: A colorimetric indicator called ABTS and a fluorogenic one known as Amplex Red. The reaction was preceded by sample irradiation with light (\u03bb = 350 nm) for 5 min, in order to produce free oxygen radicals, which could then oxidize substrates in the DNAzyme catalyzed reaction . Initialr et al. reported+\u25cf, resorufin is not a radical product, so should remain stable in the experimental conditions. The initial rates of Amplex Red oxidation were determined from the first 5 min of the reaction and the results for different systems are shown in The activity of DNAzyme on the QD surface was then examined using the oxidation reaction of the fluorogenic substrate Amplex Red. Progression of the reaction can be easily followed, since the product of the reaction\u2014resorufin\u2014exhibits strong fluorescence. Moreover, contrary to ABTSAs can be noticed, the QD-DNAzyme system exhibited higher activity towards the Amplex Red oxidation than the QD/DNAzyme mixture. It is also clear that there was no oxidation of the organic substrate in the reference systems without DNAzyme (buffer and QDs). In the case of deoxygenated solutions (5 or 15 min Ar purging and 15 min of irradiation by monochromatic light at 350 nm), the progression of resorufin production decreased dramatically for all investigated systems, in accordance with the reduced amount of generated ROS. This drop in catalytic activity was the most evident for extensively deoxygenated solutions (15 min Ar purging). The observed residual activity may be due to the production of hydroxyl radicals, together with ROS. The oxidation of Amplex Red was also performed for QD-DNAzyme systems with and without light irradiation, which produced ROS . In thisThese results prove that DNAzyme can catalyze the reaction between the indicator and ROS produced by QD light irradiation. The QD-DNAzyme system can be used for the development of assays for ROS scavengers . This reaction system can also be exploited as a novel indicator reaction for DNAzyme-related study and used for the development of new biosensors and aptasensors for DNA and other analytes. We proved that the immobilization of DNA on QDs increases the activity of DNAzyme. Furthermore, this approach can be applied, for example, in the assessment of various QDs\u2019 toxicity caused by the generation of ROS, especially in cellular conditions. The obtained results also present prospects for further research on covalent QD-DNA systems.\u22121 cm) [Studies were performed using CdTe@COOH QDs (Sigma Aldrich) and amino-modified oligonucleotide CatG4-NH2 (H2N-C6H12-5\u2032-TGGGTAGGGCGGGTTGGGAAA-3\u2032) purchased from Genomed (Poland). The oligonucleotide was HPLC-purified and its concentration was quantified by UV-Vis spectroscopy at 85 \u00b0C, with the following extinction coefficients at 260 nm: A = 15400; T = 8700; G = 11500; and C = 7400 (M\u22121 cm) . Amplex 2N-C6H12-5\u2032-TGG GTA GGG CGG GTT GGG AAA-3\u2032). This reaction was promoted by EDC and NHS addition. The first step included 15 min incubation of modified QDs with freshly prepared NHS and EDC in 10 mM Tris-HCl buffer (pH = 8.0). The concentration of DNA was optimized and the experiments were performed using 1\u201310 equivalents of CatG4 oligonucleotide, with 8 equivalents being determined as an optimal ratio of DNA to QD. The final reaction with 8 equivalents of amino-modified oligonucleotide was carried out by incubation of the reaction mixture for 45 min on a magnetic stirrer (25 \u00b0C).The total volume of the reaction medium was 200 \u00b5L. The crude product of the amine coupling of DNA to the QDs was purified using 10 kDa cut-off centrifugal filter units . For the experiments involving microscopic characterization and zeta potential measurements, the synthesis scale was 10 times higher.QD-DNA conjugates were obtained using an amine coupling reaction between carboxylic group-coated QDs (CdTe@COOH) and amino-modified oligonucleotides based on the CatG4 sequence . Prior to the visualization, the samples were applied to the copper/carbon TEM grids and allowed to evaporate. Measurements of the zeta potential and DLS experiments were conducted with a Nano ZS Zetasizer equipped with an He-Ne laser of 633 nm using 23 \u00b5M aqueous solution of QDs alone, QD-DNA conjugate, and QDs with the addition of DNA at a 1:3 molar ratio. Parameters for the above experiments were material (CdTe) RI = 1.47, water RI = 1.330, and a viscosity of water equal to 0.8872 at 25 \u00b0C.2 cell.QD and QD-DNA were characterized by UV-Vis, fluorescence, CD, and IR spectroscopy. Absorption spectra were recorded in the 220\u2013800 nm range using a Jasco V-750 spectrophotometer in 10 mm pathlength quartz cuvettes. Emission spectra were recorded with a Jasco FP-8200 spectrofluorimeter in the range of 360 to 700 nm at a 350 nm excitation wavelength, using emission and excitation slits of 5 nm with a medium sensitivity of the detector. CD spectra were recorded using a Jasco J-1500 spectropolarimeter in the spectral range from 220 to 500 nm at a rate of 200 nm/min and a number of accumulations equal to 3. The Jasco J-1500 spectropolarimeter, Jasco V-750 spectrophotometer, and FP-8200 spectrofluorimeter were equipped with Peltier temperature control accessories and all of the experiments were performed at 25 \u00b0C. IR spectra were recorded for aqueous solutions of QD and QD-DNA at 84 \u00b5M concentration using an ALPHA FT-IR spectrometer in a CaFThe electrophoresis experiment was performed in 1% agarose gel in 1 \u00d7 SB buffer (pH = 8.50) for 45 min with a 100 V (10 V/cm) voltage using a B1A electrophoresis system model . After the electrophoresis process, gel was stained with thiazole orange and visualized using UV Transiluminator .Measurements of DNAzyme activity against the generated ROS were carried out in 10 mm quartz cuvettes using the Jasco spectrofluorimeter, FP-8200 with a Peltier-type temperature accessory (irradiation and Amplex Red reaction measurements), and the Jasco V-750 spectrophotometer (ABTS reaction measurements). The samples containing QDs and other reagents were deoxygenated under argon for 0\u201315 min and then irradiated in a spectrofluorimeter at a wavelength of 350 nm with an excitation slit of 10 nm. A suitable indicator (fluorogenic\u2014Amplex Red or colorimetric\u2014ABTS) was added directly before the activity measurement started. Reactions with 5 \u00b5M Amplex Red indicator were carried out for samples containing 0.033 \u03bcM QD-DNA or QDs and 0.1 \u03bcM DNA for a reference QD/DNA system (the average ratio of DNA to QD of 3:1 was found in the QD-DNA conjugate). Reaction progression was monitored at \u03bbem = 590 nm with excitation at 560 nm. Experiments with ABTS indicator were carried out, with samples containing 0.167 \u03bcM QDs or QD-DNA, 0.5 \u03bcM DNA if needed, and 1 mM ABTS. Absorbance changes were monitored at 415 nm. In all cases, measurements were made in Tris-HCl buffer pH = 8.0 and 10 mM KCl, at a temp. of 25 \u00b0C. The concentration of hemin corresponded to the amount of G4-DNA at a 1:1 molar ratio.In total, 33 nM QD-DNAzyme and 100 nM hemin in Tris-HCl buffer pH 8.0 were mixed in a quartz cell. Amplex Red stock solution was added to this such that the final concentration was 5 \u00b5M. The cell was placed in the spectrofluorimeter and the sample was irradiated with light sequentially through 350 and 450 nm long-pass filters at regular time intervals. The fluorescence at 590 nm was monitored to follow resorufin production. In both filter modes, resorufin was excited, but only with irradiation at the 350 nm filter did QDs generate ROS. A reference experiment was carried out with Amplex Red in the absence of the QD-DNAzyme conjugate.2O2, which makes the realization of in vivo applications of DNAzymes difficult. Taking into account the possibility to control the activity of the QD-based catalyst systems, we foresee applications of this light-switchable catalyst in biocatalysis, biosensing, and the design of novel cellular assays. The obtained results also present prospects for further research on covalent QD-DNAzyme systems (additional information The QD-DNA conjugate was successfully obtained by amino coupling, using EDC and NHS as a coupling reagents. The average number of immobilized oligonucleotide molecules on the surface of the nanoparticle was three, as proved by the spectroscopic method. It was also proven that the modification of QDs with DNA had a beneficial effect on the stability of the system in aqueous solutions. The obtained QD-DNA product was used as a DNAzyme system with oxidative activity triggered by light. This approach was based on the assumption that free oxygen radicals can be generated by QDs under the influence of light. The light-directed oxidative activity of the QD-DNAzyme conjugate was confirmed by oxidation of the fluorogenic Amplex Red indicator. There is a general lack of means for controlling and triggering the enzymatic activity of peroxidase-mimicking DNAzymes, for example, cellular applications are hampered because of the harsh reaction conditions due to the presence of H"} +{"text": "Background: Glycemic variability (GV) may attribute to the pathogenesis of diabetic neuropathy. The aim of this cross-sectional study was to investigate the association between GV and distal symmetric polyneuropathy (DSPN) and cardiovascular autonomic neuropathy (CAN) in a Danish population of young adults with type 1 diabetes.Methods: Young adults between 18 and 24 years with type 1 diabetes were included in this cross-sectional study. CAN was assessed by cardiovascular autonomic reflex tests (CARTs) and heart rate variability (HRV). DSPN was assessed by light pressure, pain and vibration perception, electrochemical skin conductance, sural nerve conduction velocity (SNCV), and amplitude potential (SNAP). GV were obtained by continuous glucose monitoring including coefficient of variation (CV), SD, continuous overall net glycemic action (CONGA), and mean amplitude of glucose excursions (MAGE).Results: The study comprised 133 young adults , mean age of 22 years (SD 1.6). Unadjusted, higher CV was associated with a decreased risk of sural nerve conduction (P = 0.03), abnormal SNAP (P = 0.04) and incidents of definite CAN (P = 0.04). Likewise, higher CONGA was associated with increasing incidents of subclinical DSPN (P = 0.03), abnormal SNAP (P = 0.01), and SNCV (P = 0.02). However, both associations were not statistically significant in the fully adjusted model. Higher MAGE was associated with slightly increasing measures of HRV (P = 0.03) but only when fully adjusted. When correcting for multiple tests significance was lost. A significant association was found between HbA1c and measures of both DSPN (P < 0.02) and HRV (P < 0.03) in fully adjusted models.Conclusions: No significant associations between GV and diabetic neuropathy were found after adjusting for risk factors and multiple tests. This suggests that GV may not be a risk factor for diabetic neuropathy in young adults with type 1 diabetes. However, long-term effects of GV excursions may still play a role in the pathogenic mechanisms leading to neuropathy in later life. Distal symmetric polyneuropathy (DSPN) and cardiovascular autonomic neuropathy (CAN) are severe and common complications of type 1 diabetes . DSPN an1c to evaluate the role of glucose variability (GV): HbA1c depicts an average of blood glucose over 3 months and does not reflect incidents of hypo- and hyperglycemia on a daily basis demonstrly basis . Hence Hnd HbA1c . The intData on the association between GV and diabetic neuropathy is inconsistent, scarce, and has not previously been investigated in young adults with type 1 diabetes by applying CGM \u201319. SeveThe aim of this study was to investigate the association between modifiable glycaemic risk factors of neuropathy including GV and early and possibly reversible signs of DSPN and CAN early in the life of type 1 diabetes where preventive measures may have a substantial effect later in life. This was done in a Danish population of young adults with type 1 diabetes using the newest recommendations for assessing GV and both novel and established measures for DSPN and CAN.The study was designed as a cross-sectional observational study. The structure has been described in detail previously .In order to investigate a population with early signs of diabetic neuropathy a cohort of young adults was assessed. All participants (age between 18 and 24 years) were recruited from the outpatient clinic at Steno Diabetes Center Copenhagen, Gentofte, Denmark. Participants were included regardless of duration of type 1 diabetes. Three hundred and fifty participants received a written invitation to participate and were subsequently contacted by phone. Written informed consent was obtained prior to examination. Ethical approval was obtained from The Danish Research Ethics Committee (project id.: H-15006967) .Participants were excluded from the present study if they were not able to wear the CGM or failed to measure and log their capillary blood glucose during the 5-days of CGM-monitoring. Moreover, examination of CAN was not performed if they were treated with beta blockers.There was no basis for conducting sensible power calculations to estimate a sample size for the aim of the study, because the investigated associations have not previously been investigated in young adults with type 1 diabetes with the use of CGM. Moreover, previous studies in other patient groups have demonstrated conflicting results.DSPN was assessed and categorized according to recommendations made by the Toronto Diabetic Neuropathy Expert Group . QuestioSigns of DSPN were evaluated by established measures: Light pressure perception was assessed by applying a 10-g monofilament until it buckled to three points at the distal bilateral foot pads: just proximal to the great, third and fifth toe , 28. PaiTM performing an electrochemical skin conductance (ESC) test on the hands and feet . Age andam, USA) . Age andam, USA) . ParticiTo evaluate CAN three standard cardiovascular autonomic reflex tests (CARTs) and measures of 5 min. resting heart rate variability (HRV) were performed in a quiet examination room. HRV was assessed after 5 min of supine rest and analyzed from 5-min resting heart rate (HR). HRV indices were analyzed in time- and frequency-domain. Time-domain included the root mean square of the sum of the squares of differences between consecutive R\u2013R intervals (RMSSD) and standard deviation of normal-to-normal intervals (SDNN). Frequency-domain included low-frequency power band (LF) (0.04\u20130.15 Hz), high-frequency power band (HF) (0.15\u20130.4 Hz), total frequency power and the ratio low-frequency power/high-frequency power (LH/HF-ratio) .The 5-min resting HRV test was followed by the three CARTs including the lying-to-standing test (30:15), the deep breathing test (E:I) and Valsalva Maneuver. \u201cEarly CAN\u201d was defined as one out of the three CARTs was abnormal, \u201cdefinite CAN\u201d was defined as two or three were abnormal. Thresholds for abnormal results were age stratified . RestingIn line with the recommended criteria for examination of CAN , particiEach patient was asked to fill in the questionnaires Brief Pain Inventory (BPI) and Michigan Neuropathy Screening Instrument (MNSI) on the examination day. Participants were diagnosed with painful diabetic neuropathy if they in the BPI questionnaire answered having pain in both legs and/or both arms peripherally . A MNSI Moreover, a questionnaire considering life style factors such as smoking status and weekly amount of exercise in hours (pooled light and moderate/vigorous exercise) was filled in.The CGM sensor Enlite\u2122 was inserted into the subcutaneous tissue of the abdomen or alternatively the upper arm. Subsequently the iPro2\u2122 recorder was attached. The sensors should be worn for 5 days and the capillary finger blood glucose monitored four times daily for calibration. The software Medtronic CareLink\u2122 iPro\u2122 was used to generate data from the sensors. Participants were excluded from the study if there were not enough measurements of the capillary blood glucose to run the Medtronic CareLink\u2122 iPro\u2122 software. CV, standard deviation (SD), continuous overall net glycemic action (CONGA), and mean amplitude of glucose excursions (MAGE) were used to quantify GV . Time spBlood pressure and heart rate (HR) were measured after 10 min of rest and calculated as the mean of three consecutive measures performed with intervals of 1 min. Automated oscillometric blood pressure recorders were used .Height and weight were measured with clothes on but without shoes using a fixed rigid stadiometer and an electronic scale , respectively.All biochemical measures were analyzed from venous blood samples except for urine albumin and creatinine. Blood and urine samples were collected on the same day as the examination. The participants were non-fasting.1c was analyzed by high performance liquid chromatography on a Tosoh G7 . C-peptide was measured using a Cobas e411 . Triglycerides, HDL, and total cholesterol were analyzed by standard enzymatic colorimetry techniques on a Vitros 5600 . Serum LDL cholesterol was calculated using the Friedewald equationTriglyceride level did not exceed 4.5 mmol/l in any subject. Hence, no other LDL assessments were deemed relevant. Plasma creatinine was analyzed by two-point rate enzymatic technique. The Chronic Kidney Disease Epidemiology (CKD-EPI) equation was used to estimate eGFR or in case of skewed distributions as medians with interquartile range [IQR].Participants were excluded from the analysis of a specific test if the values were missing.1c were examined as determinants for neuropathy. The associations were assessed by logistic regression for the categorical outcomes and presented as odds ratios (OR) with 95% confidence interval (CI). Linear regression analyses were applied for continuous outcomes and presented as estimates with 95% CI. To meet model assumptions outcomes were log-transformed prior to analysis and subsequently back transformed to original scale where appropriate. To avoid small-sample bias, determinants of DSPN and CAN were not included in the analyses if the number of affected participants were <5.Both GV and HbA1c; Model 4: Adjusted as model 3 + systolic blood pressure, triglycerides, LDL cholesterol and current smoking. HR was included as a confounder in models where HRV indices were determinants.Four models of adjustments were applied: Model 1: Unadjusted; Model 2: Adjusted for age and gender; Model 3: Adjusted as model 2 + diabetes duration, BMI, exercise and HbAP = 0.05 as statistically significant and were adjusted for multiple tests by the Benjamini-Hochberg procedure and SAS, version 9.4 .1c 65.5 mmol/mol . Mean (SD) BMI was 24.7 kg/m2 (3.8) and 122 (92.4%) participants exercised regularly for an average of 9 h weekly. All participants were treated with insulin. Participant characteristics are presented in Overall, 133 young adults were included in the study. Twenty-three participants were excluded due to missing or lacking CGM-monitoring including insufficient numbers of capillary finger blood glucose monitoring. Reasons for not wearing a CGM sensor were primarily irritative/allergic reactions to the bandage patches or fear of discomfort. Mean (SD) age was 22 years (1.6), diabetes duration 11 years (5.2), and median (IQR) HbAn = 68) were diagnosed with subclinical DSPN. One patient (0.8%) had confirmed DSPN, and two (1.5%) possible DSPN. None met the criteria for probable DSPN. Prevalence estimates of symmetric abnormal SNAP and SNCV were 20.3% (n = 26) and 34.4% (n = 44), respectively. Prevalence of the composite measure of SNAP/SNCV, SNC was 48.4% (n = 62). Abnormal ESC results on feet were found in 4.5% (n = 6) and 3% (n = 4) on hands. Symmetrically abnormal VPT was detected in 0.8% (n = 1) and likewise for symmetrical neuropathy diagnosed by the BPI questionnaire. No participants had abnormal results when light touch, pain perception or MNSI questionnaire were used.The results of the prevalence of DSPN and CAN have been discussed elsewhere . In totan = 8) and early CAN in 26.9% (n = 35).Definite CAN was diagnosed in 6.1% CV was 40% and mediGreater CV was associated with a decrease in incidents of symmetric abnormalities in SNAP, SNC, and definite CAN unadjusted and when adjusted for age and gender in model 2. Only for SNC significance remained in fully adjusted models. In addition, higher CV was inversely associated to incidents of subclinical DSPN in fully adjusted models . However1c in model 3. Again, no significance persisted after correcting for multiple tests in model 3 and decreasing measures of HRV in model 4 . No signJaiswal et al. found moNyiraty et al. investigStudies investigating the association between GV and diabetic neuropathy in type 2 diabetes do not present uniform results. The number of studies investigating the association is limited and in particular studies assessing GV by CGM. However, CV in CGM was previously found to increase the risk of CAN in type 2 diabetes . Moreove1c and CAN and DSPN which to some extent shows that poor glycemic control is indeed a risk factor for diabetic neuropathy.The results of our study are pointing at GV not being a risk factor for developing CAN and DSPN. However, the study has its limitations making causal conclusions difficult. We did find a significant association between higher levels of HbAThe cross-sectional design of the study is not ideal when examining the relationship between GV and diabetic neuropathy in a causal manner. A prospective observational study design would have been more appropriate.As the study comprised 133 young adults identified with modest GV and prevalence of diabetic neuropathy there may not be enough power to extrapolate the results to the overall young population with type 1 diabetes. A larger sample size would have been beneficial in order to draw a more valid conclusion.The aim of the study was to investigate the association between GV and early possible reversible neuropathy in a young cohort. Thus, conclusions on associations are limited to type 1 diabetes patients in the age-rage of the study cohort.Novel and established methods of detecting DSPN and CAN , 35 wereCGM was used to assess GV as recommended in orderIt may be possible that more resourceful young adults chose to participate in the study after receiving the written invitation which could have caused selections bias.It is recommended that participants avoid test confounders as smoking, use of several drugs, meals, and caffeine-containing liquids before testing for CAN . The parAfter adjusting for relevant risk factors and multiple tests, no significant associations were found between GV and diabetic neuropathy in a cohort of young adults with type 1 diabetes. This finding is in line with some of the previous studies which have failed to provide consistent evidence that GV is a risk factor of development of CAN and DSPN.1c were significantly associated with both measures of DSPN and CAN which support earlier findings of high levels of HbA1c being an established and essential risk factor of diabetic neuropathy.This suggests that GV may not be a risk factor for early diabetic neuropathy in young adults with type 1 diabetes. However, the cross-sectional study approach including a relatively small sample size of young participants with modest GV and diabetic neuropathy make a strong conclusion difficult. Moreover, long-term effects of GV excursions may still play a role in the pathogenic mechanisms leading to neuropathy in later life. Increasing levels of HbAPrevious studies addressing the aim of the present project have assessed GV and DSPN and CAN by heterogenic measuring modalities hampering comparability. To improve comparability there is a need for studies using recommended measures of GV and diabetic neuropathy. Furthermore, more studies on young adults with type 1 diabetes are needed to confirm our findings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by The Danish Research Ethics Committee (project id.: H-15006967). The patients/participants provided their written informed consent to participate in this study.MC has contributed to the design of the study, acquired, analyzed, interpreted data, drafted the article, and approved the final version to be published. EH, MJ, and JF has contributed to the design of the study, analyzed, interpreted data, revised the article critically, and approved the final version to be published. CH has contributed to the design of the study, acquisition, analysis, interpretation of data, revised the article critically, and approved the final version to be published. All authors contributed to the article and approved the submitted version.JF holds stocks in Medicus Engineering. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."} +{"text": "Back and spine-related issues are frequent maladies that most people have or will experience during their lifetime. A common and sensible observation that can be made is regarding the posture of an individual. We present a new approach that combines accelerometer, gyroscope, and magnetometer sensor data in combination with permanent magnets assembled as a wearable device capable of real-time spine posture monitoring. An independent calibration of the device is required for each user. The sensor data is processed by a probabilistic classification algorithm that compares the real-time data with the calibration result, verifying whether the data point lies within regions of confidence defined by a computed threshold. An incorrect posture classification is considered if both accelerometer and magnetometer classify the posture as incorrect. A pilot trial was performed in a single adult test subject. The combination of the magnets and magnetometer greatly improved the posture classification accuracy (89%) over the accuracy obtained when only accelerometer data were used (47%). The validation of this method was based on image analysis. Different kinds of pain and dysfunction are frequent maladies that most people have or will experience during a lifetime, which are usually related to sports injuries, an accident, or other medical conditions, such as scoliosis. However, most of the upper or lower back pains are developed during the repetitive activities of day-to-day life. According to the Harvard Medical School, an efficient strategy to prevent them is as simple as paying attention to your posture ,2. MeasuThere is a wide range of posture and back shape assessment tools available, some of which are for clinical use, such as photography, goniometers, inclinometers, 2D and 3D X-ray imaging, magnetic resonance imaging, and infrared imaging. However, most of the assessments are still expensive, complicated to set up, time-consuming to operate, and need specialized training or involve radiation problems [The radiographic methods are the most used tools for the screening of the spines of patients; notably, 3D X-ray imaging produces an accurate 3D reconstruction of anatomical landmarks per vertebra. On the other hand, these tools have been shown to increase the incidence of cancer in later years, are complex to set up, are heavy, and can only be applied in laboratory environments . MoreoveThere are several solutions involving the measurement of the inclination and symmetry about the body sagittal plane . These dPhotographs can also quantify postural assessment by measuring the body angle or distance to globally assess posture both in sagittal and frontal planes, and the acquisition is cheap, fast, and easy ,14. SomeThe Spine Cop is a wearable posture correction and monitoring assistant system based on the SensorTile compact module , a BluetThis wearable device consists of a suspensory with the SensorTile located between the shoulder blades and two magnets in the region above .The area where the rigid components are placed corresponds to the areas near the cervical curve (slight forward curve in the neck)\u2014permanent magnet position\u2014and the thoracic curve (slight backward curve in the upper back)\u2014SensorTile module position\u2014since these are two points used for postural assessment .Two posture parameters can be analysed: the movement of the shoulder blades, which will induce a change in the position of the permanent magnets that will cause a magnetic signal variation in the magnetometer sensor placed in the thoracic curve ; and the angle that the thoracic tangent line makes with Earth\u2019s gravitational field , using oThe monitoring of the shoulder blades\u2019 muscles is accomplished by the magnetometer since the function of these muscles will affect the position of both N45 type magnets . We viewThe accelerometer consists of a three-axis microelectromechanical system (MEMS) accelerometer that measures the acceleration along the three sensitive axes of the sensor reaching the spine relative angle with the line of gravity in up to three planes, in up to \u00b1400 g acceleration full scale ,17. In aThe gyroscope is a sensor used to measure angular velocity and, jointly with the accelerometer (for orientation estimation in 3D space), is used to provide information on the current stance of the user . A small dimension lithium-ion battery with 100 mAh of capacity is used to ensure the comfort of the user.The posture scoring will be calculated based on experimentally obtained thresholds for the magnetometer and accelerometer values. The integrated microcontroller will be monitoring the IMU values in real time, sending the data to the application for posture processing. For each data point transmitted to the application, a binary classification (good or bad posture) will be attributed. At the end of a Spine Cop session, a global posture score will be computed based on all individual scores.A probabilistic algorithm was implemented to classify the user posture. A calibration procedure is performed first, during which the user is asked to perform a correct posture, which is registered. Afterwards, the device data are compared with the calibration result, and the posture classification is performed.As the user can only be asked to perform a good posture for calibration, the determination of an optimal threshold for good and bad posture is not possible. Therefore, the thresholds for determining the regions where accelerometer and magnetometer data indicate good and bad posture are statistically estimated.This estimation is done by acquiring a set of values while the user is performing different poses. These sets of values are then fitted to a Gaussian distribution, returning a mean vector and a covariance matrix, which can then be used to define an arbitrary region of confidence (95% was chosen for this case), within which the posture is considered to be good. When the user posture wanders away from these regions , the system classifies the user posture as incorrect.An independent calibration procedure is required for each individual user; however, the method for calculating these thresholds is the same for all users.Magnetometer and accelerometer data were extracted from the SensorTile system to assess, through the classification algorithm, the posture of the user. Both of these sensors provide a three-element vector with values corresponding to the acceleration and magnetic field in the three spatial components, respectively.i while keeping a good posture, and the values measured for each position are recorded into a set of values During the calibration procedure, the user is asked to assume a set of positions Good posture is defined as the correct skeletal alignment of the cervical, thoracic, and lumbar curves and musculoskeletal balance. The test subject was asked, during the calibration procedure, to assume a good posture, requiring the ear, shoulder top, hip, knee, and the ankle to line up vertically on standing stance, when seen from the side .Both arms downRight arm upLeft arm upBoth arms upThese different arm positions allow the classification algorithm to account for arm and hand movement during the usage of the device, thus allowing it to perform a correct posture classification even while the user is doing arm and hand movements . The device was calibrated while the user was standing up and sitting down . A photo of the various calibration positions is presented in For this work, eight calibration positions were chosen, with user keeping a straight back while having:i dataset estimated by The set of values The device was designed with the intention of being capable to be used by any adult male or female. However, different people will exhibit different body behaviors, leading to different thresholding limits. Therefore, it is of interest that the posture thresholds can be computed in a singular manner using the data acquired during calibration.X, Y, and Z. These data are fitted to a Gaussian distribution, which will present equiprobability surfaces that can be described by ellipsoids in a three-dimensional space. However, the signal in one spatial component is co-dependent on the signal measured in the remaining components . Transforming the system of coordinates into the principal components of the data , this ellipse can be more easily expressed. Thus, the trivariate Gaussian distribution presents, in the principal component space, an elliptical shape that can be expressed as (6)\u03c4i=Nc used in the calibration procedure is 0.95.For this work, the i calibration sets and returns a score of the user posture. The score is given as the percentage of that time interval the user kept a correct posture .The usage of the magnet/magnetometer combination is expected to increase the presented device classification performance, especially in cases in which the back is not perpendicular to the ground, but is still kept straight.For this test, we test one particular case, in which the user tilts his back while keeping it straight (keeping a correct posture through the entirety of the test). It would then be expected, ideally, that the posture would be classified as correct for the whole duration of the test.If only the accelerometer was used, it would be expected that the posture would be wrongly classified during the test, as it only compares the back angle with the gravitic earth field.In the crooked back test, the user was asked to repeatedly alternate between having his back in a straight and crooked position, while the computed posture state was monitored.The angle of the user\u2019s back A and B) and a video was recorded of the user bending his back into a correct and incorrect posture, while the position of the markers was recorded using the Kinovea image analysis software , an estimation of the back actual angle can be performed, thus allowing the classification to be performed correctly within a greater variety of positions.A single test was run, and the classification was run using only accelerometer data, and both accelerometer and magnetometer data, thus allowing the assessment of how much the additional magnetometer data can improve the performance of our classification algorithm .The classification algorithm results for both devices are shown in It was observed that when the classification algorithm used both accelerometer and magnetometer data, it achieved an accuracy of 85%, much higher than the case where only accelerometer data is used, with 47% accuracy.This confirms that the usage of both values greatly improves the device performance over the usage of accelerometer data only, especially when the user performs movements or poses that are different from just normal standing or sitting .Both the magnetometer and accelerometer data were used in the classification. For the purpose of validation, we considered the user to have a posture different than the calibrated one when the back angle falls below 160The expected and obtained classifications results agree with each other and yielThe device presented in this paper is not directly comparable with the other devices reported in the literature, as our device employs a probabilistic method, and other devices have either not considered the correctness of the measured posture or direcThe main advantage of our device is in its compactness, requiring just contact with two points of the back when compared with other devices which require carrying large and heavy data conversion and wireless modules , and theFurthermore, the usage of the magnet to provide information about the posture of the cervical portion of the spine provides a simplification in the device design, leading to lower power consumption and production cost.The performed study has potential limitations, specifically in what concerns the number of tested subjects and calibration of the device with a correct posture.Regarding the first point, only one test subject was thoroughly tested by the presented studies above. As is, there are not enough data to statistically confirm that the device functions correctly for the whole demographic for whom it has been designed (adults).As for the second point, the standard for correct posture was taken from literature and the To overcome these limitations, a greater test subject ensemble should be recruited and a statistical study conducted. To obtain an anatomically accurate assessment of the device performance, future tests should be conducted by a medical professional, specifically one with knowledge of orthopedics or ergonomics (or a combination of both).We developed a wearable posture monitoring system based on the SensorTile module using a combination of sensor data with permanent magnets to assess the user\u2019s back position.Through a statistical algorithm, the classification of the posture was performed and validated.The usage of magnetometer data in combination with accelerometer data displayed an improved performance (85% accuracy) when compared with using the accelerometer data alone (47%). Moreover, in a test where the user alternated between a good back position and a crooked one, an accuracy of 89% was obtained.For future work, a more thorough validation process, with posture positions anatomically verified by a physician will be performed. Furthermore, a real-time monitoring for a day-long period of time is also planned."} +{"text": "Mesenchymal stromal cells (MSC) in bone marrow have been shown to be radioresistant, which is related to pronounced DNA repair mechanisms. Intraoperative radiotherapy (IORT) during breast-conserving surgery for early breast cancer is an innovative technique applying low energy x\u2011ray to the tumor bed immediately after removal of the tumor. IORT is considered to reduce the risk of local tumor recurrence by directly targeting cells of the tumor bed and altering the local microenvironment. Aim of this study was to investigate whether IORT affects the outgrowth potential of breast adipose tissue-derived MSC (bASC) as part of the tumor bed.After surgical tumor resection, biopsies of the tumor bed were taken before (pre IORT) and after IORT (post IORT) and processed applying well-established protocols for ASC isolation and characterization.In all, 95% of pre IORT tumor bed samples yielded persistently outgrowing bASC with typical ASC characteristics: fibroblastoid morphology, proliferation, adipogenic and osteogenic differentiation and ASC surface marker expression. However, none of the post IORT samples yielded persistent outgrowth of bASC.After breast-conserving surgery, approximately 90% of local recurrences emerge in close proximity to the initial tumor bed, potentially reflecting a\u00a0significant contribution of the tumor bed to relapse. Our data show that IORT, besides the proven effect on breast cancer cells, efficiently modifies the tumor environment by having an impact on tumor bed bASC. This effect on tumor bed stromal cells might contribute to reduce the risk of tumor relapse and metastases.The online version of this article (10.1007/s00066-020-01586-z) contains supplementary material, which is available to authorized users. Breast cancer is the most frequent malignant tumor in women. Beside surgical, chemotherapeutic and receptor-targeted therapy, local radiotherapy completes the mainstays of treatment. While postoperative whole breast radiotherapy remains standard in patients undergoing breast-conserving surgery, intraoperative radiotherapy (IORT) is increasingly implemented in clinical routine. This risk-adapted concept uses low energy x\u2011rays applied during surgery directly after excision of the tumor . The ideseed and soil theory, the wound healing process after surgery is likely to provide favorable growth conditions not only for the healthy tissue, but also for residual tumor clusters [As supported by the clusters . Subsequclusters . IORT coclusters . Furtherclusters . In the clusters , 6.Mesenchymal stromal cells (MSC), as a\u00a0potential part of the tumor bed stroma, comprise a\u00a0heterogeneous population of multipotent stem/stromal cells that can be isolated from a\u00a0variety of different tissues including adipose tissue . Due to In allogeneic bone marrow transplant setting, stromal cells remain host-derived irrespective of the condition regime intensity . This suThe aim of this work was to analyze whether IORT affects the outgrowth potential of bASC, indicative for an effect on the tumor bed stroma. Biopsies of breast adipose tissue were harvested in patients with IORT before and after IORT and in control patients without IORT. Outgrowing cells were characterized against MSC criteria.A\u00a0total of 20\u00a0breast cancer patients undergoing breast-conserving surgery with (study collective) and 21\u00a0without (control collective) IORT were recruited after written informed consent was obtained. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. In women of the study collective, tumor bed biopsies were taken before (pre) and after (post) IORT.IORT was performed according to the TARGIT\u2011A protocol : The IntHistological subtypes and molecular phenotype of were comparable for IORT and controls (not shown).2. ASC growth rate was monitored recording cell number at every passage by calculating cell doublings (CD) and doubling time (DT):Twenty-four hours after biopsy, breast ASC (bASC) were isolated using an established method . BrieflyTo define multipotent MSCs, the International Society for Cell and Gene Therapy has suggested a\u00a0characteristic combination of functional properties and the specific surface marker phenotype. The hallmarks include plastic adherence, trilineage differentiation capability into adipocytes, chondrocytes and osteoblasts as well as the characteristic surface molecular marker expression, defined by the absence of hematopoietic markers and the presence of CD73, CD90, and CD105 . After p1, cells were subjected to differentiation assays. Immunophenotyping was performed after p2. Growth curves were calculated in p1 and p2.Osteogenic and adipogenic differentiation was performed as described previously using os5 cells/tube. FcR blocking reagent (Miltenyi Biotech) was added and incubated for 10\u202fmin before adding the titrated volume of antibody (see table S1). Unstained cells served as negative control. Cells were incubated 20\u202fmin, followed by two washes with PBS. Finally, Sytox Blue was added to exclude dead cells and cells were analyzed using a\u00a0FACS Canto\u00a0II analyzer running FACS Diva software (Becton Dickinson).Multicolor immunophenotyping was also performed as described before . Brieflyt-test, as applicable.Analysis was performed using GraphPad Prism\u00a07. Significance testing was done using two-way analysis of variance (ANOVA) or MSCs were isolated using collagenase digestion from breast tissue biopsies pre and post IORT (controls: pre and post sentinel lymph node resection). The sample weight was comparable with 0.45\u202f\u00b1\u20090.37 for pre non-IORT, 0.37\u202f\u00b1\u20090.26 post non-IORT and 0.56\u202f\u00b1\u20090.44 and 0.54\u202f\u00b1\u20090.29\u202fg for pre and post IORT, respectively. In all, 95% of the pre IORT samples and 57% and 66% of the pre and post non-IORT samples yielded MSC cultures, growing beyond passage\u00a02 Table\u00a0. HoweverDifferentiation assays and immunophenotyping were performed to verify the MSC-like nature of bASCs. Nearly 90% of the presamples underwent adipogenic differentiation, whereas from the post-non-IORT samples only 55% showed adipogenic differentiation properties Table\u00a0b. All bAThe immune phenotype of bASC at p2 was highly typical for ASC and corresponded to the ISCT proposed positive and negative markers to distinguish MSCs from other cells , 13. As Our results indicate that IORT entirely abolishes the adhesion and proliferation potential of bASC, suggesting radiosensitivity of bASC, at least in situ. Since IORT is performed with 20\u202fGy prescribed to the applicator surface in a\u00a0single session, supporting data that doses higher than 10\u202fGy radiosensitize MSC , 14. TheDespite the nearly high isolation success rate, pre IORT samples showed decelerated proliferation compared to non-IORT controls. Different subtypes or molecular phenotypes between the study and control cohort cannot explain these differences between pre IORT and pre/post non-IORT. The lower mean breast size in non-IORT women (exclusion criterion for IORT) and the likely different breast composition with a\u00a0higher proportion of connective tissue, which was often observed in non-IORT samples, may explain the differing success rates. Although we tried to avoid thermocoagulation during biopsy collection, it is inevitable during breast-conserving surgery depending of the degree of bleeding. Nevertheless, within the groups there was no difference, so that we consider this effect as negligible.Our data on osteogenic and adipogenic differentiation potential suggest that within the 30\u202fmin period after resecting the tumor and closing, the wound affects the differentiation\u2014but not the outgrowth and proliferation\u2014properties of bASC. Possibly the surgical stress induced (pro)inflammatory priming may has affected differentiation, previously shown to affect the balance between osteo- and adipogenic differentiation . At the Growth, morphology, differentiation, and immune phenotypic characterization documented that bASC fulfilled criteria of MSCs. IORT ultimately abolished the outgrowth capacity of bASC, indicating bASC radiosensitivity in situ. These data support the notion that IORT exerts an ablative effect within the tumor bed, not only affecting potential residual tumor cells, but also the tumor bed. By this, the IORT concept of localized radiation targeting residual tumor cells in the tumor bed to reduce the risk of relapse appears to be validated with respect to tumor bed-derived bASC. Our data may aid in explaining the efficiency of IORT concerning local recurrences and overall survival as stated in the follow-up of the TARGIT\u00a0A trial . They maIt was beyond the scope of this study to assess the effect of radiation of expanded MSC , 11, 14 After breast-conserving surgery, approximately 90% of local recurrences emerge in close proximity to the initial tumor bed, potentially reflecting a\u00a0significant contribution of the tumor bed to relapse. The goal of intraoperative radiotherapy (IORT) is to target remaining tumor cells by a\u00a0locally concentrated dose of radiation while preserving healthy tissue. Our results indicate that IORT, besides the proven effect on breast cancer cells, entirely abolishes the adhesion and proliferation potential of bASC, suggesting radiosensitivity of bASC at doses of 20\u202fGy. This might add to reduce the risk of tumor relapse and metastases.Table S1. Antibodies used for multicolor immunophenotyping"} +{"text": "Social media have emerged as increasingly popular means and environments for information gathering and propagation. This vigorous growth of social media contributed not only to a pandemic (fast-spreading and far-reaching) of rumors and misinformation, but also to an urgent need for text-based rumor detection strategies. To speed up the detection of misinformation, traditional rumor detection methods based on hand-crafted feature selection need to be replaced by automatic artificial intelligence (AI) approaches. AI decision making systems require to provide explanations in order to assure users of their trustworthiness. Inspired by the thriving development of generative adversarial networks (GANs) on text applications, we propose a GAN-based layered model for rumor detection with explanations. To demonstrate the universality of the proposed approach, we demonstrate its benefits on a gene classification with mutation detection case study. Similarly to the rumor detection, the gene classification can also be formulated as a text-based classification problem. Unlike fake news detection that needs a previously collected verified news database, our model provides explanations in rumor detection based on tweet-level texts only without referring to a verified news database. The layered structure of both generative and discriminative models contributes to the outstanding performance. The layered generators produce rumors by intelligently inserting controversial information in non-rumors, and force the layered discriminators to detect detailed glitches and deduce exactly which parts in the sentence are problematic. On average, in the rumor detection task, our proposed model outperforms state-of-the-art baselines on PHEME dataset by GANs as one of the most powerful generative models estimate generative models via an adversarial training process2. Real-valued generative models have found applications in image and video generation. However, GANs face challenges when the goal is to generate sequences of discrete tokens such as text4. Given the discrete nature of text, backpropagating the gradient from the discriminator to the generator becomes infeasible5. Training instability is a common problem of GANs, especially those with discrete settings. Unlike image generation, the autoregressive property in text generation exacerbates the training instability since the loss from discriminator is only observed after a sentence has been generated completely5. To remedy some of these difficulties, several AI approaches 9, reinforcement learning (RL)10) have been proposed12. For instance, the Gumble-softmax uses a reparameterization trick and softmax calculation to approximate the undifferentiable sampling operation on the generator output, which allows the model to perform backward propagation as well as provide discrete outputs approximating to actual values. GANs with Gumbel-softmax take the first step to generate very short sequences of small vocabulary7. WGAN method for discrete data directly calculates Wasserstein divergence between discrete labels and generator\u2019s output as the criterion of discriminator. As a result, WGAN models can update parameters to learn the distribution of discrete data and produce some short sentences in character-level9. As a result, generating natural language-level sentences is still non-trivial. GANs with RL can skirt the problem of information loss in the data conversion by modeling text generation as a sequence of decisions and update the generator with reward function. Comparing to previous methods, RL can help GANs generate interpretable text closer to natural language4. In addition to the recent development in GAN-based text generation, discriminator-oriented GAN-style approaches are proposed for detection and classification applications, such as rumor detection13. Differently from the original generator-oriented GANs, discriminator-oriented GAN-based models take real data (instead of noise) as the input to the generator. Fundamentally, the detector may get high performance through the adversarial training technique. Current adversarial training strategies improve the robustness against adversarial samples. However, these methods lead to reduction of accuracy when the input samples are clean14.Sequential synthetic data generation such as generating text and images that are indistinguishable to human eyes have become an important problem in the era of artificial intelligence (AI). Generative models, e.g., variational autoencoders (VAEs)16. The convenient and fast-spreading nature of micro-blogs fosters the emergence of various rumors. Social media rumors / misinformation / fake news are major concerns especially during major events, such as the global rise of COVID-19 and the U.S. presidential election. Some of the coronavirus rumors have been verified later to be very dangerous false claims, e.g., \u201cthose that suggest drinking bleach cures the illness\u201d17 have made social media companies such as Facebook to find more effective solutions18. Commercial giants, government authorities, and academic researchers take great effort in diminishing the negative impacts of rumors19. Rumor detection has been formulated into a binary classification problem by a lot of researchers. Traditional approaches based on hand-crafted features describe the distribution of rumors21. However, early works depending on hand-crafted features require heavy engineering skills. More recently, with the rise of deep learning architectures, deep neural network (DNN)-based methods extract and learn features automatically, and achieve significantly high accuracies on rumor detection22. Generative models have also been used to improve the performance of rumor detectors13, and formulate multi-task rumor classification systems23 to realize rumor detection, tracking, stance and veracity classification. However, binary rumor classification lacks explanation since it only provides a binary result without expressing which parts of a sentence could be the source of the problem. The majority of the literature defines rumors as \u201can item of circulating information whose veracity status is yet to be verified at the time of posting\u201d24. Providing explanations is challenging for detectors working on unverified rumors. Comparably, fake news is more well-studied, as it has a verified veracity. Attribute information, linguistic features, and semantic meaning of post25 and/or comments26 have been used to provide explainability for fake news detection. A verified news database has to be established for these approaches. However, for rumor detection, sometimes a decision has to be made based on the current tweet only. Text-level models with explanations that recognize rumors by feature extraction should be developed to tackle this problem.Social media and micro-blogging have become increasingly popular28. Since essentially a gene sequence is of textual nature, we can process a genetic sequence as text. Gene mutation detection looks for abnormal places in a gene sequence29. Hence, we propose to solve this problem by using a natural language processing-based mutation detection model. When comparing a gene sequence with a natural language sequence, we observe that the mutations in genetic sequences represent abnormalities that makes the sequence do not fit well compared to other sequences from a biological perspective. The known genetic mutation detection and classification problem has been effectively explored in the literature, while the unknown mutation detection and classification has remained as a harder problem in both medical and machine learning fields. To detect unknown mutations and classify them, we propose a GAN-based framework that maintains a high performance level while facing unseen data with unknown patterns and providing explainability capabilities.Gene classification and mutation detection usually work with textual-gene data and also relate to a broad range of real-world applications, such as gene-disease association, genetic disorder prediction, gene expression classification, and gene selection. Machine learning-based classification and prediction tools have been proposed to solve these genetic problems4 to train the layered generators. In contrast to prior works, we adopt a RL approach in our framework because by combining the GAN and RL algorithmic strategies the framework can produce textural representations with higher quality and balance the adversarial training. The training instability of long sentence generation is lowered by selectively replacing words in the sentence. We solve the per time step error attribution difficulty by word-level generation and evaluation. We show that our model outperforms the baselines in terms of addressing the degraded accuracy problem with clean samples only.In this work, we propose a GAN-based layered framework that overcomes the afore-mentioned technical difficulties and provides solutions to (1) text-level rumor detection with explanations and (2) gene classification with mutation detection. In terms of solving the technical difficulties, our model keeps the ability of discriminating between real-world and generated samples, and also serves as a discriminator-oriented model that classifies real-world and generated fake samples. We overcome the infeasibility of propagating the gradient from discriminator back to the generator by applying policy gradient similar to SeqGANOur GAN-based framework consists of a layered generative model and a layered discriminative model. The generative model generates high-quality sequences by first intelligently selecting items to be replaced, then choosing appropriate substitutes to replace those items. The discriminative model provides classification output with explanations. For example, in the gene classification and mutation detection task, the generative model mutates part of the genetic sequence and then the discriminative model classifies this genetic sequence and tells which genes are mutated. The major contributions of this work are: (1) this work delivers an explainable rumor detection without requiring a verified news database. Rumors could stay unverified for a long period of time because of information insufficiency. Providing explanations of which words in the sentence are problematic is critical especially when there is no verified fact. When a verified news database is achievable, our model is capable to realize fake news detection with minor modifications. (2) Our model is a powerful textural mutation detection framework. We demonstrate the mutation detection power by applying our proposed model to the task of gene classification with mutation detection. Our model accurately identifies tokens in the gene sequences that are exibiting mutations, and classifies mutated gene sequences with high precision. (3) The layered structure of our proposed model avoids the function mixture and boosts the performance. We have verified that using one layer to realize two functions either in generative or discriminative model causes function mixture and hurts the performance.24, usually emerge when there are influential events and spread rapidly with the rise of social media. Far-reaching and fast-spreading rumors can cause serious consequences, for example, they are growing threats to the democratic process30. Rumor detection suffers from the limitation of datasets scale and the uncertain nature of rumors makes the early-detection and classification with explanation challenging. In this section, the proposed discriminator-oriented GAN framework utilizes the layered generative model to generate augmented rumor dataset, and uses Rumors, defined as \u201citems of circulating information whose veracity status is yet to be verified at the time of posting\u201dR), or non-rumor (N), and generated data in PHEME\u2019 are all labeled as R since we would like our R/N). In real world applications, the\u00a0original clean dataset is available all the time. However, the modified or adversarial data that contains different patterns are not always accessible. Models like LSTM and CNN do not have generalization ability and usually perform worse facing adversarial input. Generative models such as GANs are more robust. In VAE-LSTM and VAE-CNN, we first pre-train VAEs, then LSTM and CNN are trained under latent representations of pre-trained VAEs. Under the first evaluation principle, our model and the variation of our model with LSTM cells outperform all baselines in terms of both macro-f1 and accuracy. Accuracy is not sufficient when the test data are not balanced, hence macro-f1 is provided for comprehensive comparison. Under the first evaluation principle, the robustness and generalization ability of our model are shown by comparing with baselines under PHEME+PHEME\u2019. Our model reaches the highest values in both versions of PHEME+PHEME\u2019 and the variation of our model with LSTM cells follows as the second best. Under leave-one-out (L) principle , our proposed model and the variation achieve the highest macro-f1 scores in all cases. These results confirm the rumor detection ability of the proposed layered structure under new, out-of-domain data. Adversarial training of baselines improves generalization and robustness under PHEME+PHEME\u2019, but hurts the performance under clean data as expected. Although our model and the variation are trained adversarially, they achieve the highest macro-f1 under clean data PHEME. The results confirm that our model outperforms the baselines in terms of addressing\u00a0the accuracy reduction problem.Table mentclass2pt{minimTable A component for decision explanation is realized by 32 and frequently suffer from small-scale datasets. The difference between misinformation, disinformation, fake news, and rumor is not well-defined and the labeling in these tasks is sometimes ambiguous and imprecise. In this work, we specifically refer rumor as a piece of information whose veracity is not verified, and its label in detection task is rumor (R)/non-rumor (N). With the consideration of veracity status, we refer facts as true (T) and false statements as false (F). Furthermore, we refer purely human-written statements as real (E) and machine-generated statements as fake (K). In the previous detection section, we do binary classification in rumor detection task. Our generative model replaces parts of a sequence and due to the uncertain nature of rumors, we label the generated (modified) rumors as R, and non-rumor in original dataset as N to emphasize the purpose of filtering out non-rumor in real-world applications. However, with real / fake and true/false labeling in misinformation or fake news classification, the labeling should be precise and 2-class labeling is not sufficient anymore for the generated (modified) sequences. Specifically, if an input sequence is labeled as Y, its modified version is labeled as Y. In what follows, we perform the following experiments: (1) rumor classification with PHEME again using 4-class labels: R, N, T, F, E, K, Misinformation, disinformation, fake news, and rumor classifications have been studied in the literatureR, N, Experimental results of PHEME (4-class) are shown in Table 33, which includes both real/fake and true/false data. In real/fake task, models differentiate between purely human-written statements and machine-generated statements, while in true/false task, models are required to identify true statements and false claims. We augment the original dataset (denoted as FMG) with our GAN-generated data (denoted as FMG\u2019) and train several models with the augmented dataset (denoted as FMG+FMG\u2019). Similarly in PHEME (4-class) experiments, we find that models trained with augmented FMG+FMG\u2019 achieve higher performance on original FMG as shown in Table Besides rumor detection, we apply our framework in misinformation and fake news detection tasks using a fake news dataset (FMG)Examples of error cases of our model in rumor detection task are presented in Table 34. Since our proposed framework demonstrates very good results for sequential / textural data (as shown in previous sections), next, we adopt a textural representation36 of gene sequences and investigate a gene mutation phenomenon. Note that binary format representation of genetic sequences is also frequently used in the literature38. In our GAN framework, the input to the models is first encoded into a high-dimensional vector, therefore, the binary formatting does not affect the experimental results. In this experiment, we first perform a mutation in genetic sequences by the generative model, and then use Genetic sequence classifications, gene mutation detection/prediction, DNA / RNA classification all work with genetic sequences, and deep learning-based methods in the literature take sequential data as input, and output the classification resultsAP, AN, DP, DN from NN269 and In this experiment, all models are trained under NN269+NN269\u2019 (an augmented dataset) to ensure fairness, and we follow the labeling rule in misinformation/fake news detection task. When testing with NN269+NN269\u2019, there are 8 classes in total: Rumor, as a piece of circulating information without verified veracity status, is hard to detect, especially when we have to point out why it is a rumor. Misinformation, whose veracity is determined, can be detected where there exists a verified database containing information about why the misinformation is wrong. Rumor detection is a hard problem and rumor detectors in the literature usually suffer from the low accuracy. The reason for unsatisfactory performance is multi-fold: for example, rumor dataset is usually small and imbalanced. The data-driven machine learning detectors don\u2019t have sufficient high-quality data to work with, hence the data shortage causes the low or extremely imbalanced performance. Rumors usually emerge violently during emergent national or even international events and confirming the veracity of rumors can take a long time and an aggressive amount of human resource. Therefore, rumors could stay as floating and circulating pieces of information without veracity confirmed for a long time and provoke social panic, such as in the recent coronavirus breakout events. Rumors are associated with different events, so if the detector is trained with previously observed rumors on other events, the detection of current unseen rumors associated with the new event usually results in low accuracy because the patterns of the rumors are changed. Compared to the detection problem, pointing out the problematic parts of the rumors is even more difficult due to the similar reasons.We propose a framework that addresses the afore-mentioned issues. To solve the limited and imbalance data issue and the low performance problem, our proposed GAN-based framework augments the dataset by generating new rumors/misinformation/fake news and uses the augmented data to train the discriminators to achieve high accuracy. The layered generative model intelligently decides about where and how to modify the input sequences. This process injects noise in data and pushes the discriminators to learn the essential semantic and syntactic features of the rumors. Therefore, this process alleviates the impact of event-associated patterns. To provide reasonable explanations of why the sentence is potentially a rumor, we improve the discriminator in GAN to include a layered structure to (1) make the detection decision, (2) generate the explanation, and (3) provide a corresponding layered model-tuning signal to the layered generative model.Genetic sequences classification, genetic mutation detection/prediction, gene-disease association, and DNA expression classification all work with gene sequences. Machine learning-based methods such as support vector machines and deep neural networks have already been used to solve these problems. We propose and verify the applicability of our designed framework on gene classification and mutation detection in this work. The fundamental rationality comes from that the genetic sequence essentially is textual data. Since our proposed framework is aiming to take textual data as input and make classification decisions, it is reasonable to apply the framework to gene data. Mutation detection in gene data is to find the abnormal places in a gene sequence and rumor detection with explanation is to find the abnormal places in a sentence. One problem facing by gene mutation detection is that there might be some unknown patterns in the gene sequence, which is similar to the generalization problem in rumor detection: unknown patterns exist in unobserved rumors. Hence, our proposed GAN-based model can alleviate this issue by intelligently augmenting the dataset. From an algorithmic perspective, the problem of rumor detection and gene classification can be formulated as a textual sequence classification problem. . Therefore, our framework as a sequential data classification model should be applicable to both rumor and gene classification. We can learn which parts are suspicious/machine generated in a rumor, and this is no different than given a sequence, we learn which parts contain abnormal patterns. Following similar reasoning, in gene mutation detection task, our model learns which parts in a genetic sequence are abnormal. The difference is that language has intuitive semantic meanings, however, genetic sequence may have unknown hidden semantic meanings. Our goal is to investigate them both even though are different in order to provide this as an example of a methodology for interdisciplinary research and analysis.In summary, we proposed a layered text-level rumor detector and gene mutation detector with explanations based on GAN. We used the policy gradient method to effectively train the layered generators. Our proposed model outperforms the baseline models in mitigating the accuracy reduction problem, that exists in case of only clean data. We demonstrate the classification ability and generalization power of our model by comparing with multiple state-of-the-art models in both rumor detection and gene classification with mutation detection problems. On average, in the 2-class rumor detection task, our proposed model outperforms the baselines on clean dataset PHEME and enhanced dataset PHEME+PHEME\u2019 by 39. We will also investigate the dependencies between the discriminators of our model to benefit Despite the high performance in both applications, we do find a limitation of our framework. We believe our proposed framework could be beneficial to numerous textual data-based problems, such as rumor and misinformation detection, review classification for product recommendation, twitter-bot detection and tracking, false information generation and attack defense, and various genetic data-based applications. We connect the genetic data processing and the natural language processing field and provide new angles and opportunities for researchers in both fields to contribute mutually.Figure M, such as a tweet-level sentence containing M words, 40 from RL to solve the non-differentiable issue.The sequence generation task is done by the generative model: 41 are the improved versions of standard RNNs that use update gates and reset gates to resolve the vanishing gradient problem of a standard RNN. In our GRU-based encoder, the hidden state z, r, and Gated Recurrent Units (GRUs)42 to automatically search for parts of a sentence that are relevant to predicting the target word. The content vector j and the output at position t match. Alignment model a is a neural network that jointly trained with all other components. The GRU decoder takes the previous target Our encoder-decoder The generated adversarial samples Y being either a rumor R or a non-rumor N. After manipulating the sequence R since it is machine generated. The objective of a N. In the rumor detection task, a sequence R. At time step t, the state The rumor generation problem is defined as follows. Given a sequence The discriminative model and the generative model are updated alternately. The loss function of discriminative model is defined as follows:We adopt the training method in GANs to train the networks. In each epoch, the generative model and the discriminative model are updated alternately. Over-training the discriminators or the generators may result in a training failure. Thus hyper-parameters Our model contains a layered generative model, 43, a misinformation/fake news dataset FMG33, and a splice site benchmark dataset NN26944. PHEME has two versions. PHEMEv5 contains 5792 tweets related to five news, 1972 of them are rumors and 3820 of them are non-rumors. PHEMEv9 contains 6411 tweets related to nine news, 2388 of them are rumors and 4023 of them are non-rumors. The maximum sequence length in PHEME is 40, and we pad the short sequences with zero padding. FMG dataset contains two parts corresponding to a veracity detection task and a provenance classification task , respectively. Input sequences with true label in veracity classification task are verified fact and false sequences are verified false statements. Input sequences with real label in provenance classification dataset are purely human-written sentences while the fake data are generated with pre-trained language models. We set the maximum sequence length as 1024 and 512 in true/false and real/fake tasks, respectively, and we pad the short sequences with zero padding and do post truncation on the text longer than length threshold. NN269 dataset contains 13231 splice site sequences. It has 6985 acceptor splice site sequences with length of 90 nucleotides, 5643 of them are positive AP and 1324 of them are negative AN. It also has 6246 donor splice site sequences with length of 15 nucleotides, 4922 of them are positive DP and 1324 of them are negative DN.We evaluate our proposed model on a benchmark Twitter rumor detection dataset PHEMEY, then the output sequence Y but with modification. In gene mutation detection task, we follow the labeling rule described in (2), and the final classification output of our model is two-fold: AP, AN for acceptor, or DP, DN for donor. We merge the generated classes In rumor detection task, we generate a rumor/fake news/misinformation dataset denoted as PHEME\u2019 (and FMG\u2019), and then augment the original dataset with the generated sequences. Similarly, for the gene classification with mutation detection task, the proposed model generates a dataset NN269\u2019 by replacing nine characters in acceptor sequences and three characters in donor sequences. We label the generated sequences by the following rules. In rumor detection with explanation task, 1) generated rumors based on PHEME are labeled as R (rumor) in 2-class classification 13. One of the strengths of our proposed model is that under the delicate layered structure that we designed, the choice of model structure affects the results but not significantly. To showcase this ability of the layered structure, we generate a variation of the proposed model by replacing 46 pre-trained vectors. Early stopping technique is applied during training. The generated data in the rumor task are labeled as R, and we denote this dataset as PHEME\u2019. For fairness and consistency, we train baselines LSTM, CNN, VAE-LSTM, and VAE-CNN with PHEME and PHEME+PHEME\u2019. For all baselines, we use two evaluation principles: (1) hold out In the rumor detection task, we compare our model with six popular rumor detectors: RNN with LSTM cells, CNN, VAE-LSTM, VAE-CNN, a contextual embedding model with data augmenting (DATA-AUG)47. The first four baselines are trained under NN269+NN269\u2019, and tested on both NN269+NN269\u2019 and clean data NN269. We import EFFECT\u2019s results from the original work47. The architectures of baselines LSTM, CNN, VAE-LSTM, and VAE-CNN used in both tasks are defined as in Table In gene classification with mutation detection task we compare our models with five models: RNN with LSTM cells, CNN, VAE-LSTM, VAE-CNN, and the state-of-the-art splice site predictor EFFECT"} +{"text": "Objective: The pathogenicity of beta-hemolytic Streptococcus group C (GCS) in patients attending for an uncomplicated acute sore throat is unknown and it was the objective to clarify this.Design: Systematic literature review with meta-analysis. Setting Medline and Scopus were searched from inception to February 2019, with searches of reference lists, Subjects case-control studies stating prevalence of GCS in patients as well as healthy controls presented for children and adults separately. Studies including patients already treated with antibiotics and studies focused on patients with HIV, malignancy or immunosuppression were not included. Main outcome measures Pooled prevalence of GCS was compared between patients and controls using chi-square and was further explored by calculating the positive etiologic predictive value (P-EPV) showing the post-test probability of a link between a sore throat and the bacterial finding. P-EPV for GCS was compared with that for group A Streptococci (GAS) using figures from the same publications and patients.Results: Eleven studies were included. The prevalence of GCS among patients versus controls was similar in children but for adults higher in patients (11%) than in controls (5.6%) (p\u2009<\u2009.0001). The P-EPV for finding GCS in children with a sore throat was 9.3% (0.0\u201341%). The corresponding P-EPV for GCS in adults with a sore throat was 53% (36\u201367%) while the corresponding P-EPV for GAS in adults was 94% (90\u201396%).Conclusions: GCS do not seem associated with the uncomplicated acute sore throat in children but there is support for an association in adults being weaker than for GAS. A possible consequence is to ignore GCS in otherwise healthy patients at their first visit for an uncomplicated sore throat. This would enable a stronger focus on the use of modern point of care tests (POCTs) to detect GAS.Streptococcus (GCS) in patients attending for an uncomplicated acute sore throat.There is no current consensus on the pathogenicity of group C beta-hemolytic This systematic literature review concludes it is unlikely that GCS is involved in the uncomplicated sore throat in otherwise healthy children.This meta-analysis found a moderate link between GCS and the uncomplicated sore throat in adults.Streptococcus.The link in adults between GCS and the sore throat is much weaker than the corresponding link for group A beta-hemolytic Streptococci (GAS) can occasionally have significant sequelae like rheumatic fever (RF) and glomerulonephritis. The risk for RF is usually very low in most high-income countries while it may be high in low-income countries. Existing guidelines for management of patients with a sore throat usually focus on beta-hemolytic Streptococci, often specifically GAS OR \u2018streptococcus\u2019 [All Fields]) AND c[All Fields] AND (\u2018pharyngitis\u2019[MeSH Terms] OR \u2018pharyngitis\u2019[All Fields]).For Scopus the search strategy used was:streptococcus and pharyngitis) AND SUBJAREA(MULT OR AGRI OR BIOC OR IMMU OR NEUR OR PHAR OR MULT OR MEDI OR NURS OR VETE OR DENT OR HEAL) AND OR LIMIT-TO OR LIMIT-TO )TITLE-ABS-KEY ,34 was cP-EPV requires an assumed sensitivity of the test to detect the etiologic agent and an assumption on the carrier rate of GCS in patients with a sore throat ill from something else, such as a virus, versus the carrier rate of GCS in healthy controls. The latter estimation is labeled theta. A gold standard is not required to calculate P-EPV making it suitable for situations where a suitable gold standard does not exist . The senThe P-EPV was calculated for each included study as well as a summary for all studies. When calculating P-EPV for several studies combined their numbers of positive and negative test outcomes for cases and controls are first added and P-EPV is calculated using figures that are the sums from included studies. Hence, studies with larger numbers will have a greater influence on the combined P-EPV.P-EPV was also calculated for GAS as a comparison in case the same studies included in the meta-analysis also presented data for GAS. P-EPV with its 95% confidence interval was illustrated graphically for scenarios where patients statistically significantly more often harbored GCS than healthy controls.Sensitivity analysis is done if patients statistically significantly more often harbored GCS than healthy controls. The main subsets to be analyzed separately are all included studies as compared to only including studies of high quality. Comparison of the outcome between these two groups will explore to what extent the main conclusions are altered by using different selection criteria. Further subsets to be analyzed separately may be identified once all publications are analyzed.PubMed search yielded 329 publications and Scopus database resulted in 469 studies . After sStreptococci (BHS). Hence, individuals with a sore throat and no growth of BHS were not classified as a case. The definition of carriers made by Belard et\u00a0al. is also ambiguous making it difficult to properly extract data so this study was not included.The study by Belard et\u00a0al. classifiThe study by Jose et\u00a0al. was a twOf the 11 studies included in the qualitative analysis, seven were of p\u2009<\u2009.0001, chi-square) of patients had GCS while 106/1897 (5.6%) of controls were positive for GCS (-square) . The sum-square) but the -square) .p\u2009=\u2009.44, chi-square) with a P-EPV of 68% (0.0\u2013100%). P-EPV is not further explored graphically for children since the difference between patients and controls did not reach statistical significance.In total 122/3836 (3.15%) of patients had GCS while 111/3878 (2.87%) of controls were positive for GCS (-square) . The sumThere is a large variation in P-EPV between included studies . The botOnly two studies provided figures specific for SDSE. One of them was of high quality and one The variation between studies in the probability of a link between a sore throat and GAS (estimated with P-EPV) shows much less variation compared to the corresponding link for GCS compare and 4. Tp\u2009=\u2009.38, chi-square). Only one study with cases and controls presented data specific for SDSE [p\u2009=\u2009.24, chi-square) with a P-EPV of 68% . None of these alternative ways of selecting studies for inclusion results in a statistically significant difference in prevalence of CGS between ill and healthy children.The prevalence of any GCS was similar in patients and controls with 3.15 and 2.87% respectively. Keeping only studies of high quality , 79 chanfor SDSE . That stOur main finding was that there was no difference in the prevalence of GCS among children with a sore throat and healthy children when only including studies having cases and controls from the same time period and geographical area. This is reflected in that the post-test probability for a link between finding a GCS in the throat and the illness a sore throat is only 9.3%.In adults a finding of GCS indicates a 53% post-test probability for this bacterium to be associated with the sore throat. This increases to 81% if only including one high quality study presenting data for SDSE. However, confidence intervals for these estimates are very wide indicating that the association between GCS and a sore throat in adults is uncertain and significantly weaker than the corresponding 94% post-test probability, with narrow confidence intervals, seen for GAS. Our conclusion is that in the absence of proof for the clinical relevance of CGS this bacterium can also be ignored in otherwise healthy adult patients at their first visit for an uncomplicated sore throat.The best method to evaluate the clinical importance of GCS would be a meta-analysis of randomized controlled studies evaluating the effect of antibiotics in sore throat patients with a sole presence of GCS. However, the few studies available are not good enough to do this.The situation is complicated by the fact that we have carriers of GCS that may be ill from something else. Ideally, we would like to have a gold standard sifting out carriers from those actually ill from GCS. However, no such gold standard exists. An increase in antibody titers could in theory be such a gold standard. However, that has been disproven for GAS and we hExpert panels to classify each case are prone to the same misclassifications as being done in today\u2019s routine medical care. Furthermore, none of the included publications used expert panels to decide for each case if they were ill from GCS or from something else. The use of a fixed composite reference standard could theoretically have been a possibility but none of the publications found used this approach. Hence, some kind of latent class modeling is required where the accuracy of testing for presence of GCS can be evaluated based on prevailing published data without having a reference standard.Since none of the conventional methods commonly used in meta-analysis was deemed suitable for this review we decided to use a previously described latent class method using a Bayesian approach, namely P-EPV ,34 to prS. anginosus group or SDSE (large colonies). It is common that publications merging GCS and GGS presenting them as one group (not being the focus of this review) do not specify if they also include small colony forming bacteria from the S. anginosus group. [S. anginosus group or SDSE. Hence, this review focusing on GCS may reflect the clinical situation encountered by many general practitioners. However, we hope publications addressing the clinical relevance of different pathogens in sore throats as well as pathology reports in the routine health care in the future will state if a non-GAS belongs to the S. anginosus group or SDSE.One limitation is that most publications included in this review omitted information as to whether the GCS belonged to the s group. Today maAnother limitation in this review is that few publications have data from patients with a sore throat as well as healthy controls collected from the same geographical area and under the same time period. This limits the number of publications available for meta-analysis.The sensitivity analysis showed that the choice of studies included in the meta-analysis is unlikely to have made any major changes in the conclusions.Cimolai et\u00a0al. first atThe carrier rates of GCS may vary among age groups, geographical areas including climate and season. McDonald et\u00a0al. reportedMcDonald et\u00a0al. speculatThe finding that GCS is associated with a sore throat in adults should be compared with the corresponding association between GAS and the sore throat estimated from the very same patients and publications. This review showed that GCS is not involved in uncomplicated sore throat in otherwise healthy children. However, the P-EPV in adults indicated a weak support for GCS to be considered as a factor in adults with a sore throat. Other publications suggest that a sore throat caused by GCS is likely to be slightly milder and with less risk for severe complications than the corresponding illness caused by GAS. Furthermore, the scientific evidence presented in previous publications for an effect of antibiotics in patients with a sore throat caused by GCS is very weak. Hence, a possible implication of this review and other publications is that it is only relevant to consider looking for presence of GCS in immunocompromised patients with an acute sore throat. There is currently not enough evidence to conclude that GCS plays an important role in otherwise healthy patients at their first visit for an uncomplicated acute sore throat and nor that antibiotic treatment for GCS help these patients. Hence, we recommend to ignore the possibility of an uncomplicated sore throat potentially being caused by GCS, a strategy already embraced by most current guidelines for managing these patients.Following these recommendations of ignoring GCS and focusing on GAS enables relying more on robust throat swabbing and rapid POCT for presence of GAS which has a much higher sensitivity and specificity compared to clinical scoring . Moving"} +{"text": "Fusobacterium necrophorum (FN) and acute sore throat in primary healthcare (PHC).The main objective of this review was to describe and quantify the association between Streptococcus (GAS).In this systematic review and meta-analysis, we searched Scopus and PubMed for case\u2013control studies reporting the prevalence of FN in patients attending primary care for an uncomplicated acute sore throat as well as in healthy controls. Only studies published in English were considered. Publications were not included if they were case studies, or if they included patients prescribed antibiotics before the throat swab, patients with a concurrent malignant disease, on immunosuppression, having an HIV infection, or patients having another acute infection in addition to a sore throat. Inclusion criteria and methods were specified in advance and published in PROSPERO. The primary outcome was positive etiologic predictive value (P-EPV), quantifying the probability for an association between acute sore throat and findings of FN in the pharynx. For comparison, our secondary outcome was the corresponding P-EPV for group A PubMed and Scopus yielded 258 and 232 studies, respectively. Removing duplicates and screening the abstracts resulted in 53 studies subsequently read in full text. For the four studies of medium to high quality included in the meta-analysis, the cumulative P-EPV regarding FN was 64% (95% CI 33% to 83%). GAS, based on data from the same publications and patients, yielded a positive EPV of 93% (95% CI 83% to 99%).The results indicate that FN may play a role in PHC patients with an acute sore throat, but the association is much weaker compared with GAS. Fusobacterium necrophorum (FN) in patients presenting with an uncomplicated acute sore throat in primary healthcare (PHC).This is the first systematic literature review with meta-analysis using positive etiologic predictive value to quantify the clinical relevance of a finding of The positive etiologic predictive value reveals the probability for a true link between FN and the sore throat expressed as a plain percentage between 0% and 100% and it was 64% (95% CI 33% to 83%).A potential limitation is that there were only four available case\u2013control studies with low or medium risk for bias presenting the proportion of FN.Streptococcus (GAS).Fusobacterium necrophorum (FN) might cause a sore throat, particularly among adolescents and young adults.7\u20139An uncomplicated acute sore throat is a common reason for attending primary healthcare (PHC).FN is an anaerobic Gram-negative bacterium most known for causing the severe disease Lemierre\u2019s syndrome, a potentially life-threatening condition that typically begins with a sore throat and is also an established pathogen in peritonsillar abscess (PTA).10\u201312The role of FN in the sore throat has been studied in three recent reviews.This study aimed to estimate the probability for an association between FN and the uncomplicated acute sore throat in patients attending PHC, when taking into consideration the carriage rate of FN in healthy controls. A second aim was to compare the probability for FN with the same probability for an association between GAS and patients with an uncomplicated acute sore throat.No patients involved.No ethical approval was needed since only publicly available data from published articles (in which informed consent was obtained by the primary investigators) were retrieved and analysed. No personal, sensitive or confidential information was collected in the scope of this study.The review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.PubMed and SCOPUS were searched (17 September 2018) for case\u2013control studies reporting the prevalence of FN in patients with an uncomplicated acute sore throat and in healthy individuals without any signs of infection. There were no time limitations. The search terms are described in 10.1136/bmjopen-2020-042816.supp1Supplementary dataAll case\u2013control studies reporting the prevalence of FN in patients attending a PHC setting for an uncomplicated acute sore throat and in a healthy control group were included. Only studies published in English were considered. Publications were not included if they were case studies, or if they included patients prescribed antibiotics before the throat swab, patients with a concurrent malignant disease, on immunosuppression, having an HIV infection, or patients having another acute infection in addition to a sore throat.SM performed the first screening by reading titles and abstract to remove duplicates from the two search strategies and, thereafter, to remove obviously irrelevant studies such as animal studies. The remaining studies were carefully screened again reading titles and abstracts independently by SM and SP to identify studies that potentially met the inclusion criteria outlined above. SM and SP started screening sitting together in the same room discussing each publication to ensure they aligned their judgement. They then continued screening separately but had a joint discussion whenever they decided differently if a publication should be kept or removedThe full texts of these potentially eligible studies were retrieved and independently assessed for eligibility by SM and SP. Any disagreement between them over the eligibility of particular studies was resolved through discussion within the whole review team.The reference lists for each article were screened for additional articles potentially matching the inclusion criteria. Such articles were added to the list of potentially eligible studies for further assessment.SM and SP independently assessed the risk of bias in the included studies by using methodological quality characteristics . OverallA standardised, pre-piloted form was used to extract data from the included studies for assessment of methodological quality and evidence synthesis. Extracted information included study setting, definition of cases and classification of these using the Centor criteria if available, definition of healthy controls, swab method, culture or PCR method, outcomes of throat swabs and information for the assessment of the risk of bias. SM and SP extracted data independently. Discrepancies were identified and resolved through discussion.A narrative synthesis was produced for each of the included studies, structured around the study methodology, target population characteristics, outcome and the assessment of methodological quality.2 test.Studies with a healthy control group and of a medium to high methodological quality were used for the meta-analysis, where the pooled difference in prevalence of FN between cases and healthy controls was compared using \u03c7The clinical relevance of any statistical differences between symptomatic patients and healthy controls was further explored by calculating the positive etiologic predictive value (P-EPV).Using a random effects meta-analysis would have provided ORs for harbouring FN among cases compared with controls. The statistical technique we used for meta-analysis, P-EPV, has been used previouslyI2) calculated in random effect models is very unreliable when the number of included publications is smallP-EPV does not directly take into account between-study variation so it is not a random effect model. We compensate for this by presenting our outcome for each individual study as well as a sensitivity analysis where we compare the consequences of combining them differently. Furthermore, the between-study variation statistics (2) . The cum2) , table 32) (2) . The cum2) (2) , table 3When including all cases (Centor score 0\u20134) in studies with low or medium risk for bias also providing data for GAS in the very same patients, the cumulative positive EPV for a finding of GAS was 93% (95% CI 83% to 99%) , table 3This literature review and meta-analysis showed that the P-EPV for FN (detected by culture or PCR) and the uncomplicated acute sore throat was 64% (95% CI 33% to 83%). The corresponding P-EPV for GAS was 93% (95% CI 83% to 99%), based on data from the same publications and patients. When limiting the analyses to the patients with Centor score 3\u20134, the P-EPV for FN was 71% (95% CI 34% to 88%) and for GAS was 97% (95% CI 91% to 100%).A potential limitation is that there were only four available case\u2013control studies presenting the proportion of FN. However, this study is the first systematic literature review with meta-analysis using P-EPV to quantify the clinical relevance of a finding of FN in patients presenting with an acute uncomplicated sore throat in PHC. As such, it represents the current best understanding of the clinical importance of FN in patients with an uncomplicated acute sore throat.The relatively high prevalence of FN in healthy controls (7.2%) indicates that FN, at least for some patients, is a part of the normal tonsillar flora. The proportion of patients with FN was 18% for Centor score 0\u20134% and 21% for Centor score 3\u20134, but the corresponding increase for GAS was from 19% to 35% . SubsequThe P-EPV numbers indicate that FN plays a role as a pathogen in patients with an uncomplicated acute sore throat. However, compared with the results for GAS, the association between FN and the uncomplicated acute sore throat appears to be considerably weaker and only marginally higher than the P-EPV of 53% (95% CI 36% to 67%) previously found for adults harbouring Group C streptococci (GCS).The high P-EPV for a finding of GAS in a throat swab is convincing and confirms the already well-established link between GAS and sore throat.6It has been suggested that antibiotic treatment of uncomplicated acute sore throat caused by FN would be cost-effective if it reduces the incidence of Lemierre\u2019s syndrome by at least 20%.For uncomplicated acute sore throat in PHC, the CI of P-EPV for FN (33%\u201383%) is wider and lower compared with the corresponding P-EPV for GAS (83%\u201399%). The level of certainty for these CIs is deemed as high as it is based on three high quality and one medium quality study including a total of 676 cases and 439 controls. Since the lower limit for the 95% CI for FN is well above 0%, we can, with a high level of certainty, state there is an association between FN and the uncomplicated acute sore throat in PHC. However, it is weaker than the same association for GAS.We are not aware of any studies showing that antibiotic treatment has beneficial effects on the duration or severity of symptoms in an FN-associated acute sore throat. Nor do we have any evidence that antibiotic treatment to patients with an uncomplicated acute sore throat reduces the incidence of the life-threatening Lemierre\u2019s syndrome. Hence, in the absence of this evidence, we do not recommend routinely searching for FN in throat swabs or prescribing antibiotics to patients with a GAS negative uncomplicated acute sore throat in PHC. However, to our knowledge, at least one randomised controlled trial focusing on GAS-negative patients with a sore throat, and analysing the presence of FN, is underway. Hence, our current advice may in the future have to be revised.More future studies should focus on randomising patients with an uncomplicated acute sore throat and presence of only FN to treatment with antibiotics or placebo in order to assess whether the treatment is effective to reduce duration and intensity of symptoms, and, more importantly, if complications such as PTA can be prevented. The prevalence of Lemierre\u2019s syndrome is so low that any effect of antibiotics on its prevalence most likely would need to be estimated using other designs than a simple clinical trial comparing antibiotics with placebo."} +{"text": "Numerous AI applications are being developed for the management of critically ill patients , both be with AI . For theaccountability and risk mitigation, where AI amplifies human cognition while humans sustain AI [The first domain is related to stain AI . The spesense-making, where AI interacts with humans in intelligible ways while hnable AI . Rather performance augmentation, where AI embodies human skills while humans train AI [The third and final domain is train AI . Real-titrain AI . In turn"} +{"text": "Over the past few years, there has been a proliferation of artificial intelligence (AI) strategies, released by governments around the world, that seek to maximise the benefits of AI and minimise potential harms. This article provides a comparative analysis of the European Union (EU) and the United States\u2019 (US) AI strategies and considers (i) the visions of a \u2018Good AI Society\u2019 that are forwarded in key policy documents and their opportunity costs, (ii) the extent to which the implementation of each vision is living up to stated aims and (iii) the consequences that these differing visions of a \u2018Good AI Society\u2019 have for transatlantic cooperation. The article concludes by comparing the ethical desirability of each vision and identifies areas where the EU, and especially the US, need to improve in order to achieve ethical outcomes and deepen cooperation. Artificial Intelligence (AI) is a cluster of smart technologies, ranging from machine learning software, to natural language processing applications, to robotics, that has unprecedented capacity to reshape individual lives, societies and the environment How the EU and the US conceptualise a \u2018Good AI Society\u2019 and the opportunity costs associated with each approach.(ii)The extent to which the implementation of each vision is living up to stated aims.(iii)The consequences that these differing visions of a \u2018Good AI Society\u2019 have for transatlantic cooperation.Having clarified how we use the expression \u2018Good AI Society\u2019, and in light of the gap identified in the literature, in the following pages we compare the development of the governance strategies of these two governments, structuring our analysis around three points:In Sects.\u00a0\u201caccountability or transparency as guiding ethical values.In May 2016, the EU released its first document addressing the issue of AI governance: a draft report, by the European Parliament\u2019s Committee on Legal Affairs, entitled \u2018Civil Law Rules on Robotics\u2019, which called for a coordinated European approach that would employ a mix of hard and soft laws to guard against possible risks. While this report began to address many of the ethical and social issues associated with the development and use of AI, Cath et al. highlighBoosting the EU\u2019s technological and industrial capacity across the economy, in both the private and public sectors.Preparing for the changes brought about by AI through anticipating market change, modernising education and training and adapting social protection systems.Ensuring that there is an appropriate legal and ethical framework that is in line with the EU\u2019s values.Since the 2016 JURI report, the focus on AI by EU policymakers has increased significantly. In April 2018, 25 European countries signed a Declaration of Cooperation on Artificial Intelligence,In June 2018, the EU Commission appointed the High-Level Expert Group on AI (HLEG),Most recently, the European Commission released the draft Artificial Intelligence Act (2021), which proposes a risk-based approach to regulating AI and outlines four categories of risk: unacceptable, high-risk, limited risk and minimal/no risk. Systems deemed to be of unacceptable risk will be prohibited, including cases of social scoring and subliminally manipulative systems (Title II). High-risk AI includes systems which are safety-critical components and those that pose specific risks to fundamental rights. For these systems, specific obligations for providers, importers, distributers, users and authorised representatives are outlined (Title III). Limited risk systems are those that interact with humans, are used for biometric categorisation or generate manipulative content (e.g. deepfakes); these systems have specific transparency requirements (Title IV). For systems which are not high risk, voluntary codes of conduct are encouraged (Title IX). Violating this regulation would lead to fines of up to 6% of global turnover or 30 million euros (Title X).EU AI policy that has\u00a0emerged since 2018 addresses many of the key drawbacks highlighted in Cath et al. together with the European Investment Fund (EIF) pledged \u20ac150 million to support AI companies across Europe released the US\u2019s first reports focusing specifically on AI: \u2018Preparing for the Future of Artificial Intelligence\u2019 and the \u2018National Artificial Intelligence Research and Development Strategic Plan\u2019. These reports defined the government\u2019s role in the development of AI as a facilitator of innovation and a minimalist regulator and outlined how federal R&D investments would guide the \u201clong term transformational impact of AI\u201d. In their assessment of these policy documents, Cath et al. stressedDriving technological breakthroughs in AI to promote scientific discovery, economic competitiveness and national security.Developing appropriate technical standards and reducing barriers to the safe testing and deployment of AI in order to enable the creation and adoption.Training American workers with the skills to prepare them for jobs of the future.Fostering public trust and confidence in AI technologies and protect civil liberties, privacy and American values.Promoting an international environment that supports research and opens markets for American AI industries, while protecting the US\u2019s technological advantage, including AI technologies from competitors.The Trump administration continued the laissez-faire approach to AI laid out in these documents, and initially went further by stating that it had no intention of developing a national plan. This position was criticised on account of its failure to stimulate investment, nurture talent and minimise harms Knight, , and thePolicy documents released since flesh out these five principles. In November 2020, as Trump\u2019s own administration was coming to a close, the White House released guidance for government agencies proposing new AI regulations for the private sector, which centres around three themes: limiting regulatory overreach; ensuring public engagement; and promoting trustworthy AI that is fair, transparent and safe , an independent commission set up to review advances in AI to address the national security needs of the US. In its final report, the NSCAI concluded that \u201c[t]he United should invest what it takes to maintain its innovation leadership, to responsibly use AI to defend free people and free societies, and to advance the frontiers of science for the benefit of all humanity\u201d , the executive order considers privacy as separate from \u2018American Values\u2019, and other policy documents also define them differently. External protectionism contrasts with internal classical liberalism, with these inconsistencies raising questions about how unified the American approach to the governance of AI is within the federal government, especially compared to the more coherent value-set that underpins the EU\u2019s \u2018Good AI Society\u2019 vision. As we will discuss in Sect.\u00a0\u201cFinally, it should be stressed that \u2018American values\u2019 are ill-defined. The website for Trump\u2019s AI initiative seems to define \u2018American values\u2019 as \u201cfreedom, guarantees of human rights, the rule of law, stability in our institutions, rights to privacy, respect for intellectual property, and opportunities to all to pursue their dreams\u201d . Despite these developments, some commentators argue that the government is not doing enough to stimulate the AI ecosystem . However, the extent to which such programmes can resolve the longer-term societal changes that AI might cause should be questioned, particularly in regard to the less-educated parts of the population. In 2018, the US was ranked 9th out of 25 advanced economies in terms of readiness for automation, with vocational technical training considered inadequate \u2014an international and multi-stakeholder initiative to guide the responsible development and use of AI\u2014due to a fear that the ethics focus would hinder innovation, it joined, like the EU and others, in May 2020. This was predominantly an attempt to counter the perceived threat of China and ensure that AI develops in line with \u2018American values\u2019 globally. Shortly after, in September 2020, the US took an active step at promoting defence cooperation through establishing the AI Partnership for Defense. This initiative seeks to share lessons, improve interoperability and increase data sharing amongst allies. Importantly, the initiative includes many European countries, such as Denmark and France, but excludes others, including Germany and the Netherlands . Other international organisations are also trying to promote international and agreement on AI, such as UNESCO, whose draft Recommendation on the Ethics of AI has been provisionally accepted by member states . Indeed, the EU\u2019s desire for digital sovereignty, which largely centres on curtailing American technology companies, is in direct conflict with the US agenda for \u2018digital free trade\u2019 and low barriers to entry for its corporations. It seems unlikely that these underlying positions will shift, given fundamental differences in areas such as digital trade which predate the Trump administration and the introduction of new ones (e.g. the draft AI Act).\u00a0This enforcement will undoubtedly be imperfect, but the measures in place will effectively guide good behaviours and penalise the worst offenders.This is not to say that the EU\u2019s vision cannot be improved upon; the inclusion of group privacy protections and more explicit measures to curtail the potential environmental harms of AI would help in achieving ethical outcomes through the improved protection and promotion of fundamental values. More generally, the EU\u2019s regulatory framework also needs to be accompanied by stronger measures that promote responsible innovation to guarantee ethical outcomes. The EU\u2019s relative lack of investment and leading technology companies has a knock-on effect into other areas, such as the attraction of AI talent.The laissez-faire path taken by the US at a domestic level is more ethically questionable. It has largely placed the governance of AI in the hands of the private sector, leaving significant scope for organisations to prioritise their own interests over that of citizens. Whilst some of the measures outlined in the National AI Initiative Act appear to be a promising step in the right direction, an emphasis on ethical and regulatory measures is largely secondary to further strengthening R&D to improve competitiveness. Without government regulatory oversight, there is a significant risk of harm through ethics washing or shopping, with the effectiveness of private sector principles questionable (Hagendorff, One continued risk for both the EU and US, if new measures are not introduced, is internal fragmentation. The benefits and risks associated with AI and data-driven technologies are already being spread unevenly across both the EU and the US, on account of different regulatory protections and opportunities from AI that are afforded to citizens. Such a fractious implementation is problematic as it will lead to uneven enforcement and unequal protection of the rights of citizens. This undermines the ability to maintain and project a consistent vision of a \u2018Good AI Society\u2019 from a governance perspective and raises ethical questions over whom is left unprotected by fractious regulations.Whilst not irreconcilable, and despite the change of administration in the US, it is unlikely that these two visions will coalesce around deep transatlantic cooperation. In fact, as the EU\u2019s digital sovereignty agenda continues to develop, increased friction with the US and its technology companies may be anticipated. This is not to say that deeper transatlantic cooperation is impossible. For instance, if relations between China and both the US and the EU continue to deteriorate, then it is possible that more substantive engagement in areas such as defence will materialise between them. Nonetheless, it would be desirable for closer alignment to instead\u00a0emerge through the US turning its statements about ethical AI into reality. For example, the statement by the Federal Trade Commission that it will consider enforcing against unfair or deceptive AI could provide a strong starting point for other agencies to announce their own measures, but this alone is inadequate. Enforceable AI regulations, meaningful anti-trust measures and a cooperative international environment, both with allies and competitors, are all necessary for a truly \u2018Good AI Society\u2019 to materialise for all those who are part of it. How these developments play out in the next few years will have a significant influence on the development and deployment of AI and the protections that are afforded to citizens in the United States, Europe and elsewhere\u00a0."} +{"text": "Carya cathayensis, an important economic nut tree, is narrowly endemic to eastern China in the wild. The complete cp genome of C. cathayensis was sequenced with NGS using an Illumina HiSeq2500, analyzed, and compared to its closely related species. The cp genome is 160,825 bp in length with an overall GC content of 36.13%, presenting a quadripartite structure comprising a large single copy , a small single copy , and a pair of inverted repeats . The genome contains 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. A total of 252 simple sequence repeats (SSRs) and 55 long repeats were identified. Gene selective pressure analysis showed that seven genes were possibly under positive selection compared with the other Juglandaceae species. Phylogenetic relationships of 46 species inferred that Juglandaceae is monophyletic, and that C. cathayensis is sister to Carya kweichowensis and Carya illinoinensis. The genome comparison revealed that there is a wide variability of the junction sites, and there is higher divergence in the noncoding regions than in coding regions. These results suggest a great potential in phylogenetic research. The newly characterized cp genome of C. cathayensis provides valuable information for further studies of this economically important species. Carya, belonging to the family Juglandaceae, comprises ~18 species and 4 varieties, which are distributed in the temperate and tropical regions of East Asia and eastern North America [Carya species from East Asia and eastern North America are phylogenetically separated [The genus America . Carya separated , while tmatK, rbcL-atpB, rpoC1, rps16, trnH-psbA, and trnL-F) and nuclear (ITS and phyA) DNA markers have been used for the phylogenetic study of the genus Carya. These nuclear genes were identified by ortholog screening, cloning, and sequencing; however, these methods can be costly and time-consuming. Compared with the nuclear genome, the chloroplast (cp) genome is an excellent alternative owing to its small size (75\u2013250 Kb) [C kweichowensis [C. cathayensis [C. illinoinensis (NBCI accession number: NC_041449.1) have been published, and the publication of more cp genomes of Carya species will facilitate the identification of genetic variations via sequence comparison, providing new insights into the evolutionary history and interspecific relationships among Carya species.Nuclear and plastid DNAs are the basics for phylogenetic reconstruction; the single- or low-copy nuclear genes are most suitable for systematic analyses . Until n\u2013250 Kb) , easily all size 5\u2013250 Kb \u2013250 Kb) ,9,10,11.howensis , C. cathhayensis , and C. C. cathayensis (Chinese hickory) is naturally distributed in moist valleys at altitudes of 500\u20131200 m in Zhejiang, Jiangxi, and Anhui Provinces, China. Because of its high nutritional and economic values, C. cathayensis has been widely cultivated in Zhejiang Province, China [C. cathayensis is an important economic nut tree and is vulnerable to abiotic factors [C. cathayensis populations has become an urgent task. The nuclear genome and cp genome of C. cathayensis have been released [C. cathayensis is essential for the development of conservation and breeding strategies.e, China . C. cath factors ,16, suggreleased ,17, althC. cathayensis and explore the utility of this new genomic resource and relationship with that of other Carya species. These results will lay the foundation for future phylogenetic and structural diversity studies of Carya.In this study, we present the whole plastome sequence of C. cathayensis were collected from the nursery of Zhejiang A&F University and stored immediately at \u221280 \u2103. Total genomic DNA was isolated from the leaves using a modified CTAB method [The young green leaves of B method . After ehttp://www.bioinformatics.babraham.ac.uk/proje-cts/fastqc/, accessed on 8 September 2021). High-quality clean reads were obtained by removing the adapters and low-quality reads from the raw data using Trimmomatic (version 0.35) [C. cathayensis cp genome was assembled using the SPAdes pipeline [Cyclocarya paliurus cp genome as the reference (NCBI accession number: NC_034315).Quality control for the raw sequencing data was carried out using the package FastQC (version 0.11.8. Available online: on 0.35) . The C. C. cathayensis cp genome annotation was performed via the CpGAVAS pipeline [C. cathayensis genome was deposited to GenBank under accession number MN892516. The circular gene map was visualized in OGDRAWv1.2. Available online: http://ogdraw.mpimp-golm.mpg.de/, accessed on 12 September 2021). Relative synonymous codon usage (RSCU) was determined by CodonW version 1.4.4. Available online: http://codonw.sourceforge.net/, accessed on 15 September 2021).pipeline . The annhttps://webblast.ipk-gatersleben.de/misa/, accessed on 21 September 2021) [REPuter ,23 was uer 2021) was applJuglandaceae species, a Bayesian inference (BI) tree was inferred using protocols suggested by [To determine the phylogenetic relationships among ested by . An aligested by ,29 with C. cathayensis cp genome to that of five related species including C. kweichowensis, C. illioninensis, C. paliurus, Juglans cathayensis, and Platycarya strobilacea. The shuffle-LAGAN mode was used in mVISTA [Quercus variabilis as the reference. The sequences were initially aligned using the MAFFT online version [Ka) to synonymous (Ks) substitutions (Ka/Ks) in protein-coding genes were determined by KaKs_Calculator.The online tool Irscope was empln mVISTA , with th version ,29, the C. cathayensis is 160,825 bp in length, with a GC content of 36.13%. The genome assembly had an average read coverage of higher than 700\u00d7. The synteny was identified by comparing the C. cathayensis cp genome to the reference region, one small single copy region, and two inverted repeat regions with 2 introns and the others with 1 intron. Gene rps12 of C. cathayensis has its 5\u2032-end exon situated in the LSC region and its 3\u2032-end exons located in the IR region (The genome of bp each) . The ovebp each) . A totalbp each) . SeventeB, ycf2) . In totaB, ycf2) , among wR region , Table 2C. cathayensis cp coding sequence was estimated. The results show that all genes are encoded by 26,476 codons, and the 4 most frequently used codons were AUU (isoleucine), AAA (lysine), GAA (glutamic acid), and AAU (asparagine), pertaining to 1145 (4.32%), 1066 (4.03%), 1040 (3.93%), and 1004 (3.79%) codons, respectively tree received strong support, with the support values ranging from 47 to 100. In addition, genera Betula, Corylus, and Ostrya were found to be sister to Juglans, whereas Platycarya and Cyclocarya were more closely related to Juglans are shown in LSC.rpl36, IR. rrn4.5, rrn23, and rrn16 are higher than 1, while the values of other genes are lower than 0.03. The results show that there is lower nucleotide diversity among the six Juglandaceae species.A cp genome identity analysis was performed on the six eference . This anus Carya . Gene nuJuglandaceae have undergone selection, the synonymous (Ks) and nonsynonymous (Ka) substitution rates were calculated [rps12 gene of C. cathayensis has its 5\u2032-end exon situated in the LSC region and its 3\u2032-end exons located in the IR regions , GC contents (36.2%), and annotated genes of the whole cp genome [C. cathayensis.Plant chloroplast genomes may have 63\u2013209 genes, but most are concentrated between 110 and 130, with a highly conserved composition and arrangement, including photosynthetic genes, chloroplast transcriptional expression-related genes, and some other protein-coding genes . As withtructure ,33, incl8\u201336.3%) ,13,34. I8\u201336.3%) ,34,35. G regions ; this reC. cathayensis cp genome in this study. We found that all genes are encoded by 26,476 codons, and the 4 most frequently used codons were AUU, AAA, GAA, and AAU; among these codons, A- and U-ending codons are common [rps15, rpoA, rpoB, petD, ccsA, atpI, and ycf1-2) of C. cathayensis underwent positive selection . The pla values) . Our reselection ; other gC. cathayensis plastomes is explained by the presence of highly diverse genes, LSC intermolecular recombination, and the co-occurrence of tandem repeats. This study demonstrates that there is a wide variability of the junction sites between the cp genomes of six Juglandaceae species, and there is higher divergence in the noncoding regions than in coding regions in the cp genome of C. cathayensis. The genus Quercus was polylogenetic, resulting from the embedded branches of the genera Lithocarpus and Castanea. The characterization of the C. catayensis cp genome provides valuable genetic information for the phylogenetic study and the development of conservation strategies of the genus Carya.The diversification of"} +{"text": "The problem that the conductive metal is easy to peel off during the fabrication process of the GNM THz device is mainly discussed. The photoelectric performance of the detector was tested at room temperature. The experimental results show that the sensitivity of the detector is 2.5 A/W (@ 3 THz) at room temperature.High sensitivity detection of terahertz waves can be achieved with a graphene nanomesh as grating to improve the coupling efficiency of the incident terahertz waves and using a graphene nanostructure energy gap to enhance the excitation of plasmon. Herein, the fabrication process of the FET THz detector based on the rectangular GNM (r-GNM) is designed, and the THz detector is developed, including the CVD growth and the wet-process transfer of high quality monolayer graphene films, preparation of r-GNM by electron-beam lithography and oxygen plasma etching, and the fabrication of the gate electrodes on the Si Graphene, a conjugated carbon sheet arranged in a 2D hexagonal lattice and an iAt present, terahertz technology is widely used in many areas, such as defense, medical diagnosis, security monitoring, communication technology and space exploration. The development of terahertz technology has put forward higher requirements for terahertz detectors. However, due to the inherent limitations of electron velocity, the performance of traditional microwave electronic transistors decreases rapidly as the frequency approaches to the terahertz (THz) band (>0.1 THz). It is also difficult for the infrared optical devices to have good applications at frequencies below 20 THz . The speAlthough some methods can open the band gap of graphene, such as using graphene nanoribbons and double-layer graphene or applying stress to graphene, due to a series of challenges it has not been able to achieve mass production in the existing semiconductor process ,12,13,14Based on the principles of the graphene FET terahertz detector, the pitch size and dimensions of the graphene nanomesh and structural parameters are decided and described in According to the device structure we designed, and the micro\u2013nano processing platform of the National Center for Nanoscience and Technology of China, the overall fabrication process of the device is divided into the following five steps as shown in 2 substrate. Firstly, the desired copper foil substrate was obtained with the method of chemical immersion cleaning combined with electrochemical polishing. Then, graphene crystals were grown in a single-temperature-zone CVD tube furnace. The temperature was raised in a low-pressure environment (the pressure was set at 600\u2013800 mTorr), and H2 was introduced when the temperature was raised. Under the condition of 1050 \u00b0C, the copper foil substrate was annealed for 1.5 h and maintained at this temperature, and H2 and CH4 (carbon source) were introduced for chemical reaction preparation. The reaction time was controlled within half an hour. After the graphene film was formed, the PMMA was coated as the protective layer. The thickness of the PMMA is about 200 nm when the spin speed is 4000 RPM and the concentration of the PMMA is 6%. Ferric chloride and hydrochloric acid were used as etching solutions to soak the copper foil substrate and some inorganic impurities. After copper foil etching, the PMMA/graphene was cleaned with deionized water. The selected substrate is a highly doped p-type silicon with a thickness of 525 \u03bcm. The thickness of the oxide layer above the silicon wafer is 285 nm. Before the transfer, the substrate was cleaned with deionized water and then dried with nitrogen. The PMMA/graphene was directly transferred to the substrate. At last, the PMMA was dissolved and removed in an acetone solution. In order to prevent graphene damage and avoid using ultrasound, the PMMA was removed by 80 \u00b0C water bath heating.Graphene film is prepared on copper foil (2 cm \u00d7 2 cm \u00d7 25 \u03bcm in volume) by CVD ,15,16, aGraphene film is essentially a single layer of carbon atoms. During the process design of the device, graphene damage should be avoided as far as possible. Therefore, the stripping process is chosen for electrode evaporation in order to avoid damage to the graphene caused by strong acid etching solutions in the electrode manufacturing process.However, the metal pattern formed on graphene by the stripping process is easy to fall off. The main reason is that the bonding between graphene and substrate is very low due to the van der Waals force. When the metal film is evaporated on graphene, the metal does not directly make contact with the substrate, and graphene may also act as a \u201cstripping adhesive\u201d in the stripping process to strip the metal directly. The phenomenon of metal peeling is observed obviously, especially in the location of the large-area metal pad as shown in In order to solve this problem, a process of etching graphene is added on the basis of the conventional stripping process, and then the metal evaporation stripping process is carried out twice. The optimized process is shown in Before spin-coating, the graphene substrate is pretreated and soaked in acetone at room temperature for half an hour. After that, the graphene substrate is cleaned with isopropanol and deionized water, and dried with nitrogen successively. Next, the pre-baking temperature for the substrate is set to 120 \u00b0C and the heating time is 3 min. Then, a 4% concentration of PMMA (950 k) is selected as the positive glue to spin onto the substrate with a spin-coating speed of 5000 RPM. After spin-coating, the substrate is post-baked at 150 \u00b0C for 2 min, as shown in 2. In order to improve the exposure efficiency, the electron-beam step is set at 50 nm.2/Si substrate to enhance the adhesion force. Meanwhile, the useless graphene easily fell off. Therefore, water bath heating and longer soaking time can be used to enhance the stripping effect without using ultrasound. The stripping result is shown in The same PMMA positive photoresist was used for spin-coating again. The peeling effect of the subsequent process could be enhanced by appropriately increasing the thickness of the spin-coating, as shown in The evaporated metal is Au with a thickness of 100 nm as shown in In order to obtain the large-area and uniform graphene nanogrid, the micro\u2013nano fabrication method of electron-beam lithography (EBL) and oxygen plasma etching (OPE) was used to obtain the corresponding size of graphene nanogrid structure materials. Firstly, a large-area single-layer graphene was grown by chemical vapor deposition on a copper substrate. It was then transferred onto heavily doped p-type Si substrates with a 285 nm SiO2 layer using polymethyl methacrylate (PMMA)-assisted wet-transfer techniques. The silicon wafer (substrate size 1.2 cm \u00d7 1.2 cm) with the transferred graphene film was soaked in an acetone solution for 12 h, then soaked in isopropanol, slightly washed with deionized water, and finally dried (<80 \u00b0C). Then, the PMMA with a concentration of 4% was used as a resist, and the spin-coating speed was set at 5000\u20136000 RPM (the corresponding coating thickness was about 200 nm). For different structures and sizes of GNM, the corresponding exposure dose was determined and selected for electron-beam exposure. At last, a graphene nanomesh structure was fabricated by oxygen plasma etching. The layout of the graphene channel between the source and drain electrodes is shown in SEM images of the graphene nanomesh structure after oxygen plasma etching are shown in As the device will work in THz frequency bands, the dielectric constant of the gate dielectric layer under the gate electrode should be as large as possible. For graphene that has been processed into nanostructure, the bonding ability with the dielectric layer on the surface of graphene and the question of easy damage should be considered carefully in the subsequent processes.3N4 on the surface of the graphene nanomesh as the dielectric layer. Because the combination of Si3N4 and graphene is much better than that of a silicon oxide dielectric layer created by the thermal oxidation process, the dielectric constant of Si3N4 (\u03b5 = 6.6) is much higher than that of silicon oxide (\u03b5 = 3.9), and Si3N4 has a higher polarized photo\u2013phonon frequency, which can reduce the phonon scattering of the graphene conductive channel and is conducive to the photoelectric application of graphene [3N4 CVD was the inert gas of N2 and NH3. In addition, the power source was a low-density plasma with a power of only 40 W. These factors could ensure that the damage to graphene is minimized in the manufacturing process of the dielectric layer.Therefore, plasma-enhanced chemical vapor deposition (PECVD) was chosen to deposit Sigraphene . Simulta3N4 dielectric layer is shown in 2, and the step setting was 50 nm. The fabrication of EBL is shown in 3N4 was grown by using the Si 500 D PECVD equipment (SENTECH). SiH4/N2 and NH3 were used as the gas source, and the reaction equation is:The growth process of the Si3N4 film would be about 60 nm, as shown in While the RF power, temperature, pressure and deposition time are set to 40 W, 55 \u00b0C, 50 Pa and 1 h, respectively, the thickness of the Si3N4, the graphene nanomesh was covered by silicon nitride, and the possibility of damage was reduced, as shown in After the fabrication of the dielectric layer Si2 and the step was set at 50 nm, the overlay precision could be ensured for the electron-beam direct writing exposure of the gate electrode pattern. The EBL result is shown in Using the above process, we fabricated an r-GNM FET THz device with a graphene nanomesh size of 14 nm \u00d7 60 nm, which is shown in According to the size of the silicon substrate, the PCB board is designed for the convenience of testing the electrical properties and terahertz detection of the FET device. The transfer characteristics of the Gr layer are shown in The IR-30 steady-state infrared light source (HawkEye Technologies) was used as the light source for the THz response test. The excitation source of the light source was powered by 2.5 V DC and could be regarded as a blackbody light source at a stable working state. A 3 THz band-pass filter is used behind the light source to obtain a 3 THz electromagnetic radiation wave. The BPF3.0 filter (TYDEX) was used to obtain the transmission spectrum, which is shown in VR is the hardness of high Leghorn. When the reference frequency was 10 Hz, the photovoltage signal intensity measured by the Golay GC-1P was 6.47 \u03bcW. Under this condition, the sensitivity of the Golay was 81.3 KV/W, and the output optical power oP was about 0.08 \u03bcW. The current sensitivity formula of the detector is as follows:iR is the current sensitivity of the detector, i is the detection current, and inP is the actual incident light power. The current sensitivity of the graphene terahertz FET detector is 12 mA/W under 3 THz radiation.In order to further determine the response rate of the graphene nanomesh FET terahertz detector, the standard Golay cell GC-1P terahertz detector (TYDEX) was used to calibrate the power of the terahertz radiation source by replacing the graphene nanomesh FET terahertz detector in the same position. According to the voltage sensitivity formula:2/Si substrate. Then, aiming at the problems of electrode stripping, the method of metal evaporation and photoresist stripping twice was used to realize the source and drain electrode lamination of graphene, and enhance the adhesion of the metal wire and electrodes with the substance. Next, the graphene nanomesh with a large-area and uniform size was fabricated by EBL and oxygen plasma etching. Lastly, the conventional metal evaporation stripping process was used to fabricate the gate electrode on the Si3N4 dielectric layer. In sum, the fabrication process of the terahertz detector based on the graphene nanomesh was designed and the terahertz detector was developed. The electrical characteristics of the graphene nanomesh were tested, which verified the regulating effect of the graphene nanomesh on the electronic energy gap. A test system was built to test the developed detector, suggesting that the THz detection sensitivity of the detector can reach 2.5 A/W at room temperature.Monolayer graphene was grown on copper foil by CVD, and the high-quality graphene film was obtained by wet transfer on the SiO"} +{"text": "This review aims to describe school nutrition interventions implemented in Asia and quantify their effects on school-aged children\u2019s nutritional status. We searched Web of Science, Embase, Ovid MEDLINE, Global Health, Econlit, APA PsycInfo, and Social Policy and Practice for English articles published from January 2000 to January 2021. We quantified the pooled effects of the interventions on the changes in body mass index (BMI) and body mass index z score (BAZ), overall and by type of intervention. In total, 28 articles were included for this review, of which 20 articles were multi-component interventions. Twenty-seven articles were childhood obesity studies and were included for meta-analysis. Overall, school nutrition interventions reduced school-aged children\u2019s BMI and BAZ. Multi-component interventions reduced the children\u2019s BMI and BAZ, whereas physical activity interventions reduced only BMI and nutrition education did not change BMI or BAZ. Overweight/obesity reduction interventions provided a larger effect than prevention interventions. Parental involvement and a healthy food provision did not strengthen school nutrition interventions, which may be due to an inadequate degree of implementation. These results suggested that school nutrition interventions should employ a holistic multi-component approach and ensure adequate stakeholder engagement as well as implementation to maximise the effects. Malnutrition covers various health conditions, including stunting, wasting, underweight, micronutrient deficiencies, overweight, obesity, or diet-related non-communicable diseases . MalnutrSchool nutrition interventions have been implemented in many countries across the world using various approaches to address malnutrition among children ,6. DurinThe effectiveness of school nutrition interventions may also vary by school levels due to different conditions of children\u2019s growth and development, and different food environments in primary and secondary schools ,14,15,16To date, the effectiveness of primary school nutrition interventions implemented in Asia is still unknown. This missing piece of evidence is crucial for nutrition policy decisions in Asia. This review, therefore, aims to determine the effectiveness of primary school nutrition programmes on reducing any forms of malnutrition among school-aged children in Asian countries.This systematic review was conducted following the Preferred Reporting Items for Systematic reviews and Meta-analyses Protocols (PRISMA-P) guidelines . It was The search was carried out in Web of Science, Embase, Ovid MEDLINE, Global Health, Econlit, APA PsycInfo, and Social Policy and Practice using these following search terms: population (student* or kid* or child* or pupil* or youth* or school?age*), outcome (BMI or body mass index or wast* or stunt* or overweight* or obes* or nutrition?status), and intervention (school?based intervention* or school intervention*). English articles published from January 2000 to January 2021 were included.Inclusion criteria: Population (school-aged children), intervention , outcome , and study design Exclusion criteria: Population (school-aged children enrolled in secondary schools or multiple school levels), study design and body mass index z score (BAZ) were calculated from differences in mean changes from pre-intervention to post-intervention between intervention and control groups.2 statistics. The levels of heterogeneity were rated as low (I2 = 25%), moderate (I2 = 50%) or high (I2 = 75%). Funnel plots and the Egger\u2019s test [Heterogeneity was measured using Ir\u2019s test were perp < 0.05 were considered as significant. Sensitivity analysis was carried out by excluding experimental studies having a high risk of bias and quasi-experimental studies.Random-effects models with inverse variance methods were used to pool the effect estimates. RevMan5.4 was usedSubgroup analysis was performed according to the following characteristics that were pre-specified in the review protocol: components of interventions versus not having a healthy food provision), duration of intervention (<1 year versus \u22651 year), sample size (<1000 students versus \u22651000 students), and engagement of parents (involved parents versus uninvolved parents).The PRISMA diagram of this review is shown in The selected papers were published between 2004 and 2020. These studies included 15 cluster randomized control trials (CRCTs), 10 quasi-experiments, and 3 randomized controlled trials (RCTs), which accounted for 53.6%, 35.7%, and 10.7%, respectively. They were conducted in nine countries and two territories from different regions of Asia, namely mainland China ,34,35,36Among CRCTs and RCTs, nine studies were categorised as high risk of bias mostly due to the lack of information on controlling possible bias for either the outcome measurement or from non-adherence, and selective reporting of findings ,44,45,52Although the double burden of malnutrition has been the main problem in Asia for decades, almost all interventions (27 out of 28 studies) aimed to address childhood overweight and obesity. Only one study was conducted to tackle undernutrition . Among tSix studies were carried out primarily by researchers in the health sector ,29,34,35To implement the interventions, five studies were conducted entirely by investigators ,39,40,43The majority of studies implemented multiple-component interventions ,49,50,51The sample sizes ranged from 32 to 8853 children. Among these, 21.4% were small studies (<100 students), 42.9% were medium studies (100\u20131000 students) and 32.7% were large studies (>1000 students). The duration of the interventions ranged from 8 weeks to five school years. Most interventions conducted 1-school-year programmes ,41,45,46Most studies reported either BMI or BAZ. Of the 28 studies, 24 studies were eligible for the meta-analysis. Four studies were excluded because they did not report usable forms of outcomes, e.g., no information of standard deviation (SD), standard error (SE), or 95% confidence interval (CI) ,41,46 or2 .Fifteen studies reported BMI as an outcome. The pooled effect of the interventions was a reduced school-aged children\u2019s BMI by \u22120.36 kg/m2 and \u22120.12 kg/m2 respectively, , while nutrition education did not show a significant change , kg/mctively, . The con08), See .2 and \u22120.23 kg/m2 , respectively and \u22120.29 kg/m2 , respectively , while the interventions with healthy food provision group did not show a significant reduction. Multi-component interventions were found more frequently in the interventions with healthy food provision than another group (9 out of 9 (100%) versus 12 out of 18 (67%), respectively). Twelve out of 18 interventions without healthy food provision (67%) were overweight/obesity treatment, while none was found in the subgroup of interventions with healthy food provision.Subgroup analysis for interventions with and without healthy food provision showed that the interventions without healthy food provision group reduced BMI with a reduction of \u22120.77 kg/mSubgroup analyses according to other characteristics of interest did not show significant differences between subgroups.The pooled effect of 15 studies reporting BAZ was a statistically significant reduction of \u22120.05 .Categorised by the interventions\u2019 component, the multi-component interventions significantly reduced BAZ by \u22120.07 , while the pooled effect size of single-component interventions was not statistically significant See . No signSimilar to the change of BMI, overweight/obesity treatment interventions showed greater effect in reducing BAZ than overweight/obesity prevention interventions with the reductions of \u22120.15 and \u22120.05 , respectively see . All oveThe interventions with parents\u2019 participation did not show an outstanding impact. These interventions provided a BAZ reduction of \u22120.05 , while the interventions without parents\u2019 participation had a BAZ reduction of \u22120.06 see . Also, tSubgroup analysis for interventions with and without healthy food provision showed slightly different BAZ reductions between the subgroups. The interventions without healthy food provision provided a BAZ reduction of \u22120.09 , while the interventions with healthy food provided a BAZ reduction of \u22120.04 . Nine out of 10 interventions with healthy food provision (90%) were multi-component interventions, while only 2 out of 10 interventions without healthy food provision (20%) were multi-component interventions. Overweight/obesity interventions equally belonged to interventions with and without healthy food provision subgroups.Subgroup analyses according to other characteristics of interest did not show significant differences between subgroups.2 and \u22120.39 kg/m2 , respectively, compared with the original \u22120.36 kg/m2 .The results of the sensitivity analysis indicated that there were no major changes of pooled effects after excluding studies with a high risk of bias or a quasi-experimental design, as shown in p = 0.2234).Publication bias also was not detected. Even though the funnel plot of the studies\u2019 effects on BMI was not perfectly symmetric see , the EggOur findings suggest that, in general, primary school nutrition interventions implemented in different Asian countries significantly reduced BMI and BAZ among school-aged children. However, the effectiveness varies with certain characteristics of the interventions. In terms of intervention components, multi-component interventions showed significant reductions for both BMI and BAZ, while single-component interventions showed a significant reduction only for BMI. In addition, multi-component interventions had a stronger effect than single-component interventions in reducing BMI. Among the single-component interventions, extra exercise sessions significantly reduced the BMI of the children, while nutrition education did not. In terms of intervention aim, overweight/obesity treatment provided stronger effects in reducing BMI and BAZ than overweight/obesity prevention interventions. Involving parents in the interventions did not significantly strengthen the effectiveness of the interventions. Interventions with school food improvement showed a smaller effect size than interventions without the component.School nutrition interventions were effective in reducing BMI and BAZ among children of all ages in western/high-income countries and China ,8,9,10. The results also suggested that multi-component interventions are more effective than single-component interventions, which are in line with the findings of meta-analyses from other contexts ,9,10. ThThe stronger effect found on treatment than prevention of obesity and being overweight is in accordance with the findings from other meta-analyses in children of all ages ,52. ThisThis review also found that parent involvement did not significantly increase the effectiveness of school nutrition interventions, which is not in line with the findings from other meta-analyses ,8. A revThis review found that interventions with healthy food provision significantly decreased BMI and BAZ of the children. It was also reported elsewhere that a healthy school food environment was effective in reducing students\u2019 BAZ . In addiAcross all types of intervention, multi-component school nutrition interventions are the best option that provide consistent and strongest impacts in addressing childhood overnutrition. Among single-component interventions, extra exercise sessions have the potential to be mildly effective as a standalone component, while nutrition education should be a supplementary component.Although parental involvement has been widely recognised as a promising strategy, insufficient involvement may compromise the effectiveness. To gain benefit from implementing a healthy food environment, the criteria for healthy food should comply with school nutrition standards and practice guidelines.The way primary studies reported the outcomes is important. Incomplete or unclear information restricts the ability to use the evidence. A significant proportion of studies selectively reported only certain forms of outcomes that are not comparable to the majority of literature, causing those studies to be excluded from secondary analyses. Also, not many studies provided clear information on intervention implementation , especially components related to food and physical activity environment and parental support. The lack of information compromised the usefulness of these included studies. Therefore, future evaluative studies on school nutrition interventions should provide complete information on both intervention implementation and outcomes. Existing tools such as the Template for Intervention Description and Replication (TIDieR) checklist could beAn interpretation of the findings of this review may require careful consideration due to the following limitations. Firstly, this review is restricted to English articles published in peer-reviewed journals, so some evidence published only in Asian languages might have been excluded. Secondly, a high degree of heterogeneity was detected in the pooled effect analysis. Sensitivity tests showed that there are no concerns related to study quality and study design. The school nutrition interventions are complex with variations of actors, intervention intensity, and surrounding environments. The complexity of interventions may be related to the considerable degree of heterogeneity. Thirdly, identifying factors contributing to the effects are not feasible. This is because the number of primary studies was not large enough, and the interventions\u2019 contents were not clearly described for all studies.Primary school nutrition interventions implemented in Asia are effective in reducing BMI and BAZ among school-aged children. Multi-component interventions provided promising outcomes in reducing the children\u2019s BMI and BAZ. Among single-component interventions, extra exercise has the potential to reduce BMI, but nutrition education did not lead to significant changes. Overweight/obesity reduction interventions are more effective than overweight/obesity prevention interventions potentially due to different levels of intensity. Parental involvement and a healthy food provision do not always boost the effectiveness of school nutrition interventions, especially when the implementation is not sufficient. Comprehensiveness and intensity are key factors that must be considered seriously when designing school nutrition interventions to maximise the interventions\u2019 effects. Studies assessing the impacts of school nutrition interventions should report complete information related to the interventions and outcomes to ensure their maximum benefit."} +{"text": "Faxonius rusticus from six lakes across a relative abundance gradient. We conducted six assays to measure individual specialization of behavior and used stable isotope analysis to measure individual specialization of diet. We then related both measures of individual specialization to relative abundance of F.\u00a0rusticus using linear and quadratic models. We found a unimodal relationship between intraspecific competition and individual specialization of diet for F.\u00a0rusticus, likely because some preferred resources are unavailable to specialize on at the highest densities of this well\u2010studied crayfish invader. Conversely, we found greater support for a linear relationship between individual specialization of behavior and intraspecific competition, perhaps because specialization by behavior is not inherently resource\u2010limited. Our results show that dietary and behavioral specialization may exhibit different responses to increased intraspecific competition, and demonstrate a potential technique that can be used to investigate individual specialization of diet and behavior simultaneously for the same individuals and populations.Individual specialization within populations is increasingly recognized as important in both ecology and evolution, but researchers working on intraspecific variation in behavior and diet infrequently interact. This may be because individual specialization on diet and behavior was historically difficult to investigate simultaneously on the same individuals. However, approaches like stable isotope analysis that allow hindcasting past field diets for laboratory organisms may provide opportunities to unite these areas of inquiry. Here, we tested the role of intraspecific competition on individual specialization through analysis of both behavior and diet simultaneously. We focused on intraspecific competition as a mechanism that might drive individual specialization of both diet and behavior. We conducted this study in Vilas County, Wisconsin, United States (US), using rusty crayfish Individual specialization within populations is increasingly recognized as important in both ecology and evolution, but researchers working on intraspecific variation in behavior and diet infrequently interact. Here, we tested the role of intraspecific competition on individual specialization through analysis of both behavior and diet simultaneously. We found a unimodal relationship between intraspecific competition and individual specialization of diet for populations of an invasive crayfish, but we found greater support for a linear relationship between individual specialization of behavior and intraspecific competition, perhaps because specialization by behavior is not inherently resource\u2010limited. Long\u2010term monitoring of populations of F.\u00a0rusticus in northern Wisconsin provided us with an a priori understanding of F.\u00a0rusticus relative abundances within these lakes from baited trapping following a mid\u2010summer molt to reproductively active Form I . We used male Form I crayfish because of their higher availability to collection in our lakes during mid\u2010 to late summer, but future research on this question could also use juvenile and female crayfish.Following baited trapping, we collected crayfish by hand while snorkeling for use in our behavioral and dietary analyses. Hand\u2010collection of crayfish avoids biases that may be associated with collecting crayfish by baited trapping, which selects for larger and more aggressive individuals of pelagic and littoral benthic energy pathways between these lakes. Specifically, \u03b413C is routinely used to represent dependence of consumers on either pelagic or littoral benthic energy pathways in freshwater lakes, whereas \u03b415N is used to estimate trophic position , freshwater snails have generally enriched (more positive) \u03b413C values that represent the littoral benthic primary producers they consume , and both mussels and snails provide baseline organisms with a trophic position of two that other consumers can be compared against , and Planorbidae snails from one lake (Presque Isle). We were unable to find any snails in Papoose Lake likely due to consumption by a high abundance F.\u00a0rusticus population to assess potential for differences in the baseline stable isotope ratios , a field station operated by the Center for Limnology at the University of Wisconsin, Madison, US. Crayfish were kept in separate compartments in tackle boxes during transit to isolate the crayfish from each other. We provided a shallow supply of water in the tackle boxes to keep the crayfish moist during transport to the laboratory. At TLS, the crayfish were kept in their own individual small containers (1\u00a0L) to avoid interactions. The containers were filled with water supplied from adjacent Trout Lake, and water was changed every other day. Containers were aerated by air stones connected to air pumps via tubing. We added shelter structures to these containers prior to bringing the crayfish to the laboratory. The crayfish were fed half an algae wafer per day, except when fasting prior to behavioral assays. Past research has demonstrated that 2.3F.\u00a0rusticus individuals. These behavioral assays were chosen to represent four of the five temperament traits in animal behavior: boldness, exploration, activity, and aggressiveness were all conducted in separate 5.5\u2010L experimental arenas (36\u00a0cm length\u00a0\u00d7\u00a023\u00a0cm width\u00a0\u00d7\u00a010\u00a0cm height). For each of these five assays in experimental arenas, the water was changed between each crayfish observation, and each individual was acclimated for 15\u00a0min prior to the start of the assay. We used black plastic sheeting to create a blind to obscure the observer from the crayfish.The first behavioral assay measured the activity and exploration of individuals. For this assay, we observed shelter occupancy by crayfish over 12\u00a0hr following the 1\u2010day acclimation period, as a measure of activity. Less active crayfish were anticipated to remain in shelters throughout the day, whereas more active crayfish were anticipated to explore. During the 12\u2010hr period from 8\u00a0a.m. to 8\u00a0p.m. an observer recorded whether the crayfish was within the shelter hourly.The second behavioral assay measured exploration by assessing the willingness of crayfish to explore a new area from Allequash Lake in Vilas County, Wisconsin, US, as our high\u2010quality food item (\u201csnail\u201d) and conditioned Red Oak Quercus rubra leaves collected from Presque Isle Lake as our low\u2010quality food items , while the second assessed the time it took for the crayfish to take a lower quality food item (detritus). For each of these feeding assays, the first section of the experimental arena initially contained the individual being observed, the second section of the container contained the food item, and the third section of the container was empty. We used F.\u00a0rusticus present in the third (previously empty) section of the arena. Size\u2010matched conspecifics were within a mean of 3.9\u00a0mm (\u00b1 3.87\u00a0mm SD) total carapace length of the study individuals. The clear dividers permitted the crayfish to see each other, but not to physically interact, while the study individual foraged. As in the food quality feeding assays, the divider between the study crayfish and the snail was removed after the 15\u2010min acclimation period, and the observer recorded the time until the crayfish first attempted to feed on the item. In each of the three feeding assays, we recorded the latency to feed with a maximum time of 20\u00a0min .The third feeding assay measured the boldness of the individual to feed in the presence of a conspecific individual. We replicated the snail feeding assay above, but with a size\u2010matched conspecific The sixth behavioral assay examined the aggressiveness of individuals. To test the fight or flight responses of individuals, we observed their response to a novel object moving toward them , was dissected for stable isotope analysis, after which all samples were placed in a drying oven (Fisher Scientific Isotemp 100\u00a0L Oven) at 60\u2103 for 24\u00a0hr. Dried samples were homogenized using a stainless\u2010steel mortar and pestle that was cleaned between each sample. We then used a microbalance to weigh homogenized tissue into tins . We next shipped samples to the University of California\u2010Davis Stable Isotope Facility, where they were analyzed using a PDZ Europa ANCA\u2010GSL elemental analyzer with a PDZ Europa 20\u201020 isotope ratio mass spectrometer . The sample values were corrected using laboratory reference standards, which have a long\u2010term standard deviation of 0.2\u2030 for carbon and 0.3\u2030 for nitrogen.2.513C and \u03b415N values, whereas less similar diets of individuals within a population will have more dispersed or variable \u03b413C and \u03b415N values. As stable isotopes integrate diet continuously over time for individuals (a repeated measure), large population niche widths in this case correspond to higher individual dietary specialization, whereas smaller population niche widths correspond to lower individual dietary specialization with this same measure of individual specialization when standardizing 95% confidence ellipses to the \u03b413C values of primary consumers in each lake.Stable isotopes can be used to infer individual diet specialization by a variety of measures that calculate the breadth of population niches using the correlation matrix through the vegan package and individual specialization as the area of 95% confidence ellipses from both diet and behavior measures using Base R. Linear fits could represent increasing or decreasing individual specialization in response to relative abundance. Quadratic fits were anticipated to indicate a positive unimodal relationship between individual specialization and relative abundance, although these fits could also reflect a negative unimodal relationship where individual specialization was lower at intermediate relative abundances. We then used likelihood\u2010ratio tests to compare the linear and quadratic models for both dietary and behavioral specialization to each other, as well as to a null model (intercept only), with the lmtest package exhibited relatively small 95% confidence ellipses, indicating less individual specialized by diet, and some populations exhibited relatively large 95% confidence ellipses , indicating more individual specialization by diet , whereas the third axis was marginally significant (SD\u00a0=\u00a01.01). The first axis explained 28.29% of the variance, and the second axis explained 18.42% of the variance , as well as a significant difference between the quadratic and null models . We found no significant difference between the linear and null model for this comparison . Accordingly, we interpret the quadratic model as more supported, and this model fits the data relatively well with an adjusted R2 of 0.77 and no significant difference between the quadratic and null models . Further, the quadratic model in this case fits a weakly negative relationship between behavioral specialization and intermediate relative abundances, contrary to our a priori prediction. However, we did find a significant difference between the linear and null models of behavioral specialization and relative abundance . We interpret the linear model for this relationship as more supported, and this model had an adjusted R2 of 0.44 and therefore has low individual dietary specialization. At intermediate abundances, some F.\u00a0rusticus individuals may specialize to exploit a broader selection of diet items, like primary producers or lower quality detritus, as high\u2010quality food becomes scarce. At very high abundances, F.\u00a0rusticus may altogether deplete high\u2010quality resources, resulting in a contraction of individual specialization to lower quality or less preferred food. Our unimodal relationship between F.\u00a0rusticus dietary specialization and relative abundance is highly consistent with past work on this invasive crayfish, which has found hyperabundant populations of F.\u00a0rusticus to strongly reduce the abundance and richness of benthic invertebrates such as snails in our study lakes , to other systems and similar questions ; data curation ; formal analysis (lead); methodology ; validation ; visualization (lead); writing\u2010original draft (lead); writing\u2010review & editing . Eric R. Larson: Conceptualization (supporting); data curation ; formal analysis (supporting); funding acquisition (lead); methodology ; project administration (lead); resources (lead); supervision (lead); validation ; visualization ; writing\u2010review & editing ."} +{"text": "Although the results indicate that the silyl groups have strong electronic effects on anthracene, the details of the mechanisms responsible for this have not yet been clarified. This article describes the analysis of the UV/Vis and fluorescence spectra of 9,10-bis(diisopropylsilyl)anthracene by theoretical calculations. This study reveals that \u03c0 conjugation of anthracene is extended by cooperation of \u03c3\u2013\u03c0 and \u03c3*\u2013\u03c0* conjugation between the silyl groups and anthracene. This effect increases the transition moment of the \u03c0\u2013\u03c0* transition of anthracene. As a result, the molecular extinction coefficient of the Studies on the electronic effects of silyl groups go back to the middle of the last century. The electronic effects of silyl groups were recognized by unusual accelerations of the reactions of some organosilicon compounds . Later, 1, 1La band in the UV/Vis spectrum of 1 (\u03bbmax = 399 nm (\u03b5 = 14200 mol\u20131 L cm\u20131)) shows a considerable bathochromic shift with the large molecular extinction coefficient compared with that of anthracene (\u03bbmax = 374 nm (\u03b5 = 8000 mol\u20131 L cm\u20131)). The fluorescence quantum yield of 1 (\u03a6f = 0.90 (room temperature)) is much larger than that of anthracene (\u03a6f = 0.32 (room temperature)). These results indicate that the silyl groups affect both the HOMO and LUMO. Similar results have also been reported by other groups [In 1996, we reported that silyl groups of 9,10-bis(diisopropylsilyl)anthracene 1, and relar groups ,27,28,291 at the TD-DFT B3LYP/6-31G(d) level. We found that cooperation of electronic effects of the silyl groups in the HOMO and LUMO is important in the UV/Vis and fluorescence spectra of 1.In spite of numerous studies on the UV/Vis and fluorescence spectra of silyl-substituted aromatic compounds, the effects of silyl groups have not yet been fully determined. On the other hand, studies on the UV/Vis spectra of unsubstituted aromatic compounds have been fruitful in recent years . Studies1La band of 1 at 300\u2013450 nm with that of anthracene [1 and anthracene were measured The molecular extinction coefficient of the 1B band ) is much smaller than that of anthracene ); (2) On the other hand, the molecular extinction coefficient of the 1La band ) is larger than that of anthracene (\u03bbmax = 375 nm (\u03b5 = 7200 mol\u20131 L cm\u20131)); and (3) The 1B and 1La bands of 1 show bathochromic shifts compared with those of anthracene.In our previous paper , we compthracene ,32,33,34measured . The intectively ,36,37. T1 and anthracene at 77 K ) is much larger than that of anthracene (\u03a6f = 0.34 (77 K)) [1 and anthracene could not be observed. The phosphorescence quantum yield of anthracene has been reported to be very small ) . The pho= 0.0003 ). The da1 and anthracene are shown in 1 are similar to those of anthracene. However, the HOMO of 1 clearly shows out-of-phase interaction between \u03c3(Si\u2013C) orbitals and a \u03c0 orbital of the anthracene moiety and \u03c0* orbitals of anthracene coefficient of 0.70 (1) and 3.59 eV (anthracene). The smaller energy gap of 1 is in accordance with the bathochromic shift of the 1La band compared with that of anthracene.The HOMO, HOMO\u20131, LUMO and LUMO+1 of of 0.70 . The ene1 and anthracene are 1.46 and 0.85 a.u., respectively. The large transition moment of 1 corresponds to the large molecular extinction coefficient of the 1La band of 1 and anthracene are nearly in line with the molecular extinction coefficients of the 1B bands . These results indicate that the difference of the intensity of the 1B bands could be ascribed mainly to the electron transition, although the effect of vibronic coupling could not be completely excluded.The TD-DFT calculation showed that the his band A. Both tnsitions B. Althou1B band of 1 compared with that of anthracene is ascribed to contribution of the HOMO-to-LUMO+3 transition (1B band of anthracene are \u22120.487 (HOMO\u20131-to-LUMO) and 0.516 (HOMO-to-LUMO+1), whereas those of 1 are smaller, i.e.,: \u20130.441 (HOMO\u20131-to-LUMO) and 0.501 (HOMO-to-LUMO+1). Instead, the HOMO-to-LUMO+3 transition contributes with the CI coefficient of 0.186. The HOMO-to-LUMO+3 transition of anthracene has a high energy (6.47 eV) and does not interact with the HOMO\u20131-to-LUMO (4.82 eV) and HOMO-to-LUMO+1 (4.94 eV) transitions. However, the energy level of the LUMO+3 of 1 is very low because of effective \u03c3*\u2013\u03c0* conjugation between \u03c3*(Si\u2013C) orbitals and a \u03c0* orbital of anthracene and interacts with the HOMO\u20131-to-LUMO (4.65 eV) and HOMO-to-LUMO+1 (4.87 eV) transitions. The HOMO-to-LUMO+3 transition does not have a transition moment in the direction of the long molecular axis are much smaller than those of the 1La bands , the 1Lb bands are hidden by the 1La bands in both compounds.The TD-DFT calculations also show that the 1) of 1 and anthracene were calculated at the TD-DFT B3LYP/6-31G(d) level and compared with those of the ground states (S0). The structures and structural parameters are shown in 0 and S1 states of anthracene have completely planar structures with the Dh2 symmetry. The anthracene moiety of the S0 state of 1 has a slightly bent structure with a fold angle (4.7\u00b0) between the C9\u2013C9a\u2013C1\u2013C2\u2013C3\u2013C4\u2013C4a\u2013C10 and C10\u2013C10a\u2013C5\u2013C6\u2013C7\u2013C8\u2013C8a\u2013C9 planes. The fold angle increases in the case of the S1 state (12.6\u00b0). The bent structure of the S1 state of 1 could be explained by the effects of molecular orbitals and steric hindrance. By excitation from the S0 state to the S1 state, an electron in the HOMO is transferred to the LUMO. The p orbitals at the 9,10-positions, which have the largest coefficients in the HOMO, interact with the adjacent p orbitals with the in-phase mode (peri-positions is present in the planar structure. A combination of these electronic and steric effects results in the bent structure of the anthracene moiety.The optimized structures of the excited singlet states (Sase mode . In cont1 state of 1 has another feature. The C(1)\u2013C(2), C(3)\u2013C(4), C(5)\u2013C(6) and C(7)\u2013C(8) bonds (1.405 \u00c5) and the C(9)\u2013C(9a), C(4a)\u2013C(10), C(10)\u2013C(10a) and C(8a)\u2013C(9) bonds (1.438\u20131.441 \u00c5) are longer than the corresponding bonds of the S0 state, whereas the C(2)\u2013C(3) and C(6)\u2013C(7) bonds (1.387 \u00c5) and the C(1)\u2013C(9a), C(4)\u2013C(4a), C(5)\u2013C(10a) and C(8)\u2013C(8a) bonds (1.413\u20131.414 \u00c5) of the S1 state are shorter than the corresponding bonds of the S0 state. These structural changes are due to the electron transition from the bonding (or antibonding) \u03c0 orbital of the HOMO to the antibonding (or bonding) \u03c0* orbital of the LUMO. Furthermore, the Si\u2013C(9) (1.904 \u00c5) and Si\u2013C(10) (1.900 \u00c5) bonds of the S1 state of 1 are shortened compared with the corresponding bonds of the S0 state. The shortening could be explained by \u03c3*\u2013\u03c0* conjugation between the silicon and C(9 or 10) atoms in the LUMO.The optimized structure of the S1 states of 1 and anthracene emit fluorescence and are deactivated to the S0 states according to the Franck\u2013Condon principle [1 states of 1 and anthracene calculated by using the optimized S1 structures are shown in 1 is similar to that of anthracene, lobes of the HOMO show \u03c3\u2013\u03c0 conjugation (1 is lower than that of anthracene due to \u03c3*\u2013\u03c0* conjugation. The energy gap between the HOMO and the LUMO of the S1 state of 1 (2.90 eV) is smaller than that of anthracene (3.11 eV). This result is in accord with the bathochromic shift of the fluorescence bands of 1 compared with those of anthracene is larger than that of anthracene (0.93 a.u.) by cooperation of \u03c3\u2013\u03c0 and \u03c3*\u2013\u03c0* conjugation, similar to the UV/Vis spectra (1 (\u03bc2 = 2.42 a.u.2) and anthracene (\u03bc2 = 0.86 a.u.2) are nearly proportional to the fluorescence quantum yield of 1 (\u03a6f = 0.90) and anthracene (\u03a6f = 0.32) [1 is larger than that of the electron transition from the HOMO to the LUMO of the S0 state of 1 (1.46 a.u.). The larger transition moment of the S1 state is ascribed to the shortening of the Si\u2013C bonds. The short Si\u2013C bonds lead to effective \u03c3\u2013\u03c0 and \u03c3*\u2013\u03c0* conjugation, as shown by larger lobes of the \u03c3(Si\u2013C) and \u03c3*(Si\u2013C) orbitals than those of the S0 state . Fluorescence spectra were obtained on an F-4500 fluorescence spectrophotometer . Fluorescence quantum yields were determined by using a C9920-02 absolute PL quantum yield measurement system .0 and S1 states of 1 and anthracene were optimized at the B3LYP/6-31G(d) and TD-DFT B3LYP/6-31G(d) levels, respectively, and the optimization was confirmed by frequency calculations. The results are summarized in 1 and anthracene were calculated at the TD-DFT B3LYP/6-31G(d) level by using the optimized structures. The results are summarized in All theoretical calculations were performed by using Gaussian 09 and 16 on a PRI1. Cooperation of the \u03c3\u2013\u03c0 conjugation in the HOMO and the \u03c3*\u2013\u03c0* conjugation in the LUMO leads to the bathochromic shifts of the UV/Vis and fluorescence bands. Furthermore, the decrease of the 1B band, the increase of the 1La band, and the increase of the fluorescence quantum yield are rationalized. This is the first step toward clarifying the substituent effects of silyl groups on the optical properties of silyl-substituted aromatic compounds. To develop this work, further studies on the effects of silyl groups at various substitution positions of aromatic compounds and the effects of substituents on a silyl group are necessary. These studies will be undertaken in the near future.This study reveals substituent effects of the silyl groups on the UV/Vis and fluorescence spectra of"} +{"text": "Insecticides are considered to be one of the major factors of bee decline. In this study, the potential sublethal effects of selected neonicotinoids on honey bee larvae were investigated by protein expression profiling for the first time. The total larval protein expression was investigated by 2D gel electrophoresis after exposure to the insecticides dimethoate, fenoxycarb and flupyradifurone. Protein spots whose concentrations differed significantly from the controls were sequenced and identified against known insect proteins. Although the treated larvae did not show increased mortality or an aberrant development, the proteome comparisons showed differences in the metabolism, immune response and energy supply of the bee larvae. The strongest influence was found for flupyradifurone, which activates various detoxification pathways, the immune response or tissue regeneration. Our results suggest that there may be a delayed larval development or possibly a reduced honey bee brood vitality at sublethal concentrations.Apis mellifera is globally distributed due to its beekeeping advantages and plays an important role in the global ecology and economy. In recent decades, several studies have raised concerns about bee decline. Discussed are multiple reasons such as increased pathogen pressure, malnutrition or pesticide use. Insecticides are considered to be one of the major factors. In 2013, the use of three neonicotinoids in the field was prohibited in the EU. Flupyradifurone was introduced as a potential successor; it has a comparable mode of action as the banned neonicotinoids. However, there is a limited number of studies on the effects of sublethal concentrations of flupyradifurone on honey bees. Particularly, the larval physiological response by means of protein expression has not yet been studied. Hence, the larval protein expression was investigated via 2D gel electrophoresis after following a standardised protocol to apply sublethal concentrations of the active substance (flupyradifurone 10 mg/kg diet) to larval food. The treated larvae did not show increased mortality or an aberrant development. Proteome comparisons showed clear differences concerning the larval metabolism, immune response and energy supply. Further field studies are needed to validate the in vitro results at a colony level.The western honey bee Apis mellifera is considered to be one of the world\u2019s most important farm animals, mainly due to its high importance as a pollinator, pollinating more than 70% of global crops [Varroa destructor and accompanied virus infections, habitat loss, malnutrition and an increased application of insecticides as well as other plant protection products [The western honey bee al crops ,2. Withial crops ,4,5,6. Iproducts . There aproducts . They beproducts . In 2013products . Thus, iproducts , impact products and toxiproducts , which c50 for honey bees is specified to be 1.2 \u00b5g of the active substance (a.s.)/bee [Flupyradifurone is a next-generation butenolide insecticide that belongs to the same group as neonicotinoids , acting .s.)/bee . Primari.s.)/bee , affecte.s.)/bee or alter.s.)/bee . However.s.)/bee ,21,22. T.s.)/bee establis.s.)/bee . Whilst .s.)/bee . Therefo.s.)/bee , to studApis mellifera (Buckfast) sister queens. The colonies remained at the Institute for Bee Protection at the Julius K\u00fchn Institute in Braunschweig . The experimental colonies were healthy, had a sufficient food supply and showed no symptoms of disease or increased parasitism. No medical treatments were given four months before running the experiments.In vitro experiments were performed during spring and summer 2018/19 using three queen-right colonies with n = 16 per colony; n = 48 per insecticide treatment and controls) were reared and exposed according to the protocols of the OECD Guidance Document 239 [Honey bee larvae until day six (D6) post-grafting, with a constant concentration of flupyradifurone that was dissolved in the diet. This resulted in a cumulative dose on D6, according to w/w) and the carbamate fenoxycarb were used as additional test substances, with a known toxicity in developing honey bee larvae. Previous experiments using sublethal concentrations showed no increased mortality between a water control and an acetone control, and no differences in mortality or development between the control and treatment groups. Based on the results of a previous transcriptome study [Flupyradifurone , dimethoate and fenoxycarb were not used in combination with other substances. Acetone was used to prepare the stock solution of flupyradifurone and all subsequent dilutions. The test solution in the final diet was 0.5% me study , the livTM, Benchmark). Amounts of 10 mg DTT, 5 \u00b5L PMSF and 1 \u00b5L protease inhibitor cocktail (Merck Biosciences) were added to a 1 mL lysis buffer before the homogenisation. After incubation on ice for 1 h, the samples were centrifuged and the supernatant was used for a further clean-up following the protocol of the manufacturers . Finally, the dried protein samples were dissolved in an appropriate volume of a DIGE buffer . The total protein concentration was measured in duplicate using a Pierce 660 nm Protein Assay and bovine serum albumin as a standard (50 \u00b5g/mL\u20132 mg/mL).Each honey bee larva was homogenised in a 300 \u00b5L lysis buffer using bead tubes and a bead-based homogeniser . Isoelectric focusing (IEF) was performed within a pH range of 3 to 10 using immobilised pH gradient (IPG) strips and a Protean IEF Cell (BioRad) system at 20 \u00b0C with the following cycle: 50 V for 14 h, 200 V for 1 h, 500 V for 1 h, 10,000 V for 1 h and 10,000 V for 6 h.For the proteome analysis, single protein samples were labelled with either 400 pmol Cy3 or Cy5 , respectively. Cy2 was used as an internal standard (common reference) containing equal amounts of the pooled samples. Next, 2D gels were loaded with 50 \u00b5g total protein of a mixture of each Cy3-, Cy5- and Cy2-labelled sample in a 300 \u00b5L volume of DeStreakFor the molecular weight analysis, the IPG strips were incubated for 15 min in an equilibration buffer containing 10 mg/mL DTT and equilibrated for 15 min with an equilibration buffer containing 25 mg/mL iodoacetamide. The strips were transferred onto vertical 12.5% SDS-PAGE gels and sealed with 0.5% low-melting-point agarose. The second-dimension separation was run using an Ettan DALTsix electrophoresis unit with the following parameters for six gels: 2 W for 1 h and 12 W. This continued until the dye front reached the end of the gels.Protein spots were visualised with a Typhoon 9400 fluorescence scanner at the respective wavelengths of the Cy dyes. The spot detection and the matching and quantification of the spot intensity were performed using DECODON Delta 2D software . Only spots with more than a 1.5-fold difference in density (enhanced or decreased expression) were considered for the subsequent protein analysis. All gels and protein analyses were performed with four biological replicates.n = 4) were mixed and filled with DeStreakTM Rehydration Solution to a final volume of 300 \u00b5L in the absence of Cy dyes. A protein ladder was used to estimate the molecular weights of the target proteins. The gels were silver-stained and the selected spots (see Apis mellifera were used. As fixed (f) and variable (v) modifications, the following were chosen: carbamidomethyl (f) of cysteines; oxidation (v) of methionine; and deamidated (v). The matched peptides of the identified proteins after 2D gel electrophoresis and the identification via LC/MS are summarised in A second 2D gel electrophoresis was run with 400 \u00b5g protein per gel to pick the proteins with a significant fold change between the treatment groups. Multiple lysates per treatment group (pots see were manpots see . Trypsinp < 0.05).The image analysis and spot quantification of the protein spots were performed with DECODON Delta2D 4.5.3. software (DECODON GmbH). Differences in the expression between the samples (a minimum of a 1.5-fold change between the treatment vs. the solvent control) were analysed using unpaired two-tailed Welsh\u2019s t-tests (p < 0.001) and 3-ketoacyl-CoA thiolase . In addition, major royal jelly protein 2 and 14-3-3 protein zeta were significantly downregulated.A protein analysis of 8-day-old larvae treated with sublethal concentrations of flupyradifurone resulted in 951 detectable protein spots in total. Of these, 129 spots showed differential intensities, with 22 upregulated and 107 downregulated proteins. Peptide mapping (LC/MS) identified five selected protein spots . Two of p = 0.02), 3-ketoacyl-CoA thiolase and glutathione S-transferase S1-like protein . Major royal jelly protein 2 was again downregulated (p < 0.001) after treating the larvae with sublethal concentrations . Three o = 0.02) . Within < 0.001) ; Table 2The chronic exposure of honey bee larvae to sublethal concentrations of dimethoate, fenoxycarb and flupyradifurone caused significant changes in the protein regulation (mostly downregulation) without increasing the mortality or affecting the larval development. Five proteins were identified as significantly differentially regulated; major royal jelly protein 2 was downregulated in all treatment groups. Here, we only discuss the flupyradifurone-induced larval protein changes. However, it has to be mentioned that glutathione S-transferase S1-like protein was also downregulated in imidacloprid-treated honey bees . This deTM feed to adult honey bees [Insecticides lead to oxidative stress in many organisms and, consequently, to cell or tissue damage . Naturalney bees ) and may3-Ketoacyl-CoA thiolase was downregulated in all cases of insecticide treatments in the current and an earlier study . This enSublethal doses of flupyradifurone significantly reduced the 14-3-3 protein zeta abundance although this negative trend was also found for the other two insecticides. The family of 14-3-3 proteins are highly conserved in eukaryotes, ranging from yeast to mammals ,44,45. IMajor royal jelly protein 2 was strongly downregulated after feeding the larvae with sublethal concentrations of all three insecticides. Comparative observations have been described for pyraclostrobin (fungicide)- and fipronil (insecticide)-fed nurse bees . Royal jIn summary, this protein analysis showed that dimethoate, fenoxycarb and flupyradifurone had an impact on larval metabolism and development, but without any clear pattern related to their specific mode of action. Flupyradifurone had the strongest impact on the protein change among the list of identified protein spots. The activation of different pathways initiating detoxification, the immune response or tissue regeneration in honey bee larvae might increase their metabolic rate and, therefore, the need for nutrients that are provided by carbohydrates and major royal jelly proteins . An alteFollowing the OECD Guidance Document 239 on honey bee larval toxicity tests, this study showed differences in protein abundance with regard to larval metabolism, the immune response and energy supply after feeding honey bee larvae with sublethal concentrations of three different insecticides. During the larval development, the larvae showed relatively strong effects on the protein expression, which was consistent with previous larval transcriptome data. The sublethal concentrations of the tested insecticidal substances did not cause an increase in mortality or affect the larval development . Regardi"} +{"text": "Diabetes mellitus (DM) is a chronic metabolic disease characterized by inappropriately elevated blood glucose levels. If not treated at the early stage, it can lead to complications like diabetic retinopathy (DR) and diabetic nephropathy (DN) which are often associated with severe morbidity and mortality. This study was designed to identify the prevalence of retinopathy and nephropathy in diabetic patients and also\u00a0to determine the correlation between DR\u00a0and DN. In this cross-sectional study, a total of 84 diabetic patients were included. The mean age at presentation was 54.06 \u00b1 9.85 years. Among them, 28% of patients had a duration of diabetes of < 5 years. Nearly 42% and 30% of patients had diabetes between 5-10 years, and more than 10 years respectively. At the time of presentation to us, a total of 42.8% of patients had a combination of nephropathy and retinopathy, 40.4% of patients had only retinopathy, and 16.6% of patients with only nephropathy. Among patients with nephropathy and\u00a0microalbuminuria, only 5.9% had DR ranging from mild to a moderate degree and none had severe DR. In patients with macroalbuminuria, 26.2% had moderate to severe DR. Microvascular complications are more prevalent in diabetics with disease progression. Microalbuminuria is a marker for retinopathy and these patients require ophthalmic evaluation at the earliest. Early recognition and management of these, can reduce the occurrence of complications as well as disease progression, thus reducing the related mortality. The term \u2018Diabetes Mellitus\u2019 (DM) is derived from the Greek word 'Diabetes' which means siphon to pass through and a Latin word 'Mellitus' meaning sweet . It is aThis metabolic dysregulation in diabetes also causes secondary pathophysiologic changes in multiple organ systems resulting in chronic vascular and non-vascular complications. Major complications of DM are microvascular complications such as retinopathy, neuropathy, and nephropathy which can cause severe morbidity if neglected . This stThis is a cross-sectional study done by\u00a0convenience sampling technique in patients aged 18 and more years with type \u2161 diabetes who were attending the outpatient departments of the General Medicine department attached to a tertiary care teaching hospital in southern India from January 2013 to December 2013.Diabetic patients with other associated conditions altering fundus examination findings like, those who have had ocular procedures or surgeries in the past, patients with hypertension, congestive cardiac failure, active urinary tract infection, and pregnant diabetics were excluded from the study. This study was approved by the Institute Ethics committee of Vydehi Institute of Medical Sciences and Research centre with No: VIMS/IEC/PGThesis/2012.Case definitionsPatients were considered to be having nephropathy if they have microalbuminuria or overt proteinuria based on a positive albustix test in \u22652 consecutive urine samples. A 24-hour urine sample was collected from 8 am to 8 am and was subjected to an automated urine protein analyser which employs a turbidimetric method to quantify proteinuria. DN was graded as microalbuminuria (30-300mg/24 hours), macroalbuminuria (300-3000mg/24 hours), and massive proteinuria (>3000mg/24 hour).Patients were labelled to be having DR\u00a0on the basis of fundoscopic and ocular examination done by physicians which included best-corrected visual acuity (BCVA) using Snellen\u2018s charts, colour vision, dilated fundus evaluation using direct/indirect ophthalmoscope, and slit lamp. DR was defined and graded based on the Early Treatment of Diabetic Retinopathy Study (ETDRS) classification into Mild, Moderate, Severe, and Very severe non-proliferative diabetic retinopathy (NPDR) and Proliferative diabetic retinopathy (PDR) with or without high-risk characteristics . DiabetiBlood investigations such as fasting blood sugar (FBS), postprandial blood sugar (PPBS) & Glycosylated haemoglobin (HbA1c), blood urea, and serum creatinine were measured for all patients.Statistical analysisThe quantitative data were represented as mean, median, interquartile range, and standard deviation (SD). Frequency percentage was used for categorical variables.A total of 84 diabetic patients were included in the study. The mean age at presentation of these patients was 54.06 \u00b1 9.85 years (31-90 years). Among them, 28% of patients had a duration of diabetes < 5 years and 42% had a duration of diabetes between 5-10 years, and the remaining 30% had diabetes for more than 10 years.\u00a0Among these diabetic patients, 80.9% were on oral hypoglycaemic agents (OHA) and 19.1% were on insulin therapy. Approximately 16.6% of patients had only nephropathy and 40.4% of the patients had only retinopathy. The remaining 42.8% of patients had a combination of both. Among patients with retinopathy, 38.1% of the patients had DR with diabetes mellitus with a duration of diabetes\u00a0less than 5 years, 25% of patients had retinopathy with a duration of diabetes between 5 to 10 years, and 20.2% of the patients had DR with a duration of diabetes more than 10 years. The number of patients with moderate NPDR, severe NPDR and PDR increased as the duration of DM increased , nearly 15.5% had microalbuminuria, and 9.5% had massive proteinuria. In patients with microalbuminuria, 9.5% did not have DR, 5.9% had DR ranging from mild to a moderate degree, and none had severe DR. In the group with macroalbuminuria, only 4.7% did not have DR, whereas a majority of patients (26.2%) had moderate to severe DR. Among the patients with massive albuminuria, 2.4% did not have DR, while 7.1% had moderate to severe DR. The relationship between DR and DN is shown in table 4.5%, neaWe hereby describe our experience with microvascular complications of DM and the features of disease progression with time. Studies that looked at the incidence of DR and the association between DN and DR are very limited in India. Hence, this study was done to identify the prevalence and association of DN and DR in our study population. The increasing prevalence and longer duration of diabetes is more likely to alter the disease profile in many diabetic patients, especially with a higher incidence of microvascular complications .One of the most important complications of DM is DN, which is characterized by the triad of hypertension, proteinuria, and renal impairment. To date, DN remains a major cause of morbidity and mortality in diabetics and is prevalent in 30-35% of diabetic patients globally and is associated with a 2-4 fold increased risk of death in comparison to the general population ,5. SimilIn a meta-analysis done in 2013 with 26 studies, it was found that PDR was a more specific predictor of DN . ConversUsually, DR precedes DN. However, the converse may also be possible. As DN is microvascular in origin which starts with the thickening of the glomerular basement membrane early in diabetic renal disease, this results in nodular, diffuse, and exudative glomerular lesions leading to renal glomerular hyalinization lesions. This is primarily an ischemic event and shares similar risk factors including prolonged duration of diabetes and poor glycaemic control .In our study, it was also observed that the presence of proteinuria was more in patients with retinopathy and was directly related to the severity of nephropathy. Similarly, in the Chennai Urban Rural Epidemiology Study (CURES), proteinuria was observed in nearly one-third of the patients with DR . Even stMany epidemiological studies done previously support the role of DN as one of the risk factors for the development of DR suggesting that DN patients benefit from having a regular ophthalmic evaluation. Conversely, the presence of retinopathy is also a risk indicator of DN and these patients may benefit from screening for 24-hour urine protein estimation. Early detection of DN and its treatment could reduce morbidity and mortality of such patients from other complications of DN and DR .In our study, there was a unidirectional correlation association between DR and DN which can be explained by chronological order, that is DN precedes DR. It also indicates that the level of renal impairment is proportional to the level of damage to the eye similar to studies observed before ,12. All A major limitation of this study was as only type \u2161 diabetics were included, the other types of DM were not studied. However Intensive diabetic control leads to a reduction in the development and progression of all diabetic complications.In the present study, there is a direct correlation between DN and DR. Microalbuminuria or macroproteinuria is a marker for retinopathy and these patients require ophthalmic evaluation at the earliest. It is recommended to screen all newly detected diabetic patients for diabetic retinopathy and microalbuminuria. Also, regular follow-ups are advised with increasing duration of diabetes. A good glycaemic control and regular follow-ups can reduce the occurrence as well as the progression of the disease, thus reducing the related mortality and morbidity."} +{"text": "Diabetic retinopathy is a major microvascular complication of diabetes, and may progress to sight-threatening stages causing blindness with a consequent decrease in their quality of life. This study aimed to find out the prevalence of blindness among patients with type II diabetes mellitus attending the Outpatient Department of Ophthalmology of a tertiary care hospital.A descriptive cross-sectional study was conducted among patients with type II diabetes mellitus presenting to the Outpatient Department of Ophthalmology of a tertiary care centre from 2 August 2021 to 30 June 2022 after receiving ethical approval from the Institutional Review Committee (Reference number: 74/2021). Diabetic patients underwent detailed eye examination including vision, slit lamp biomicroscopy examination, and fundus evaluation with full pupil dilation. Convenience sampling method was used. Point estimate and 95% Confidence Interval were calculated.Among 449 type II diabetic patients, blindness was seen in 17 (3.79%) patients. Among them, 1 (5.88%) had severe non-proliferative diabetic retinopathy, 3 (17.65%) had proliferative diabetic retinopathy and 8 (47.06%) had severe diabetic macular oedema.The prevalence of blindness among patients with type II diabetes mellitus was less than in other studies conducted in similar settings. Screening and timely management of diabetic retinopathy could reduce the prevalence of blindness due to diabetic retinopathy. World Health Organization (WHO) has estimated that diabetic retinopathy is responsible for 4.8% of the 37 million cases of blindness throughout the world.2 So, with this increasing trend of diabetes in Nepal, complications due to Diabetes mellitus (DM) are likely to increase including visual impairment and blindness but there is no existing data regarding this situation.According to WHO, there were 436,000 diabetic cases in Nepal in the year 2000 but by the year 2030, it will increase to 1,328,000 cases.This study aimed to estimate the prevalence of blindness among patients with type II diabetes mellitus attending the Outpatient Department of Ophthalmology of a tertiary care centre.A descriptive cross-sectional study was conducted among patients with type II diabetes mellitus presenting to the Outpatient Department of Ophthalmology of Dhulikhel Hospital from 2 August 2021 to 30 June 2022 after obtaining ethical approval from the Institutional Review Committee (Reference number: 74/2021). Informed written consent was obtained from all the participants of this study. All the type II diabetic patients, of any age and gender, presented within the study period were included in the study. Patients with mature cataracts, patients having anterior segment and other posterior segment diseases were excluded from the study. Convenience sampling was done. The sample size was calculated using the following formula.Where,n= minimum required sample sizeZ= 1.96 at 95% Confidence Interval (CI)p= prevalence taken as 50% for maximum sample sizeq= 1-pe= margin of error of, 5%The minimum sample size calculated was 385. However, 449 patients were included in the study.3 According to the best corrected visual acuity of the patients, blindness was graded according to the WHO classification of blindness.4Demographicdetailsand information regarding diabetes mellitus and treatment received were obtained from each participant. All the patients underwent eye examinations: distant visual acuity and near vision assessment followed by retinoscopy. The best corrected visual acuity (BCVA) was noted for each participant. Then, a slit lamp biomicroscopy examination was performed to detect any anterior segment and posterior segment abnormalities. They underwent full dilatation of the pupil with a combination of 0.8% tropicamide and 5% phenylephrine eye drop followed by fundus evaluation then status and grade of diabetic retinopathy were recorded. A fundus photograph was taken and recorded with diabetic retinopathy whenever feasible. Classification of diabetic retinopathy was done according to the International Classification of Diabetic Retinopathy Scale.Data were collected and entered in Microsoft Excel 2011 and analysed in IBM SPSS Statistics 11.5. Point estimate and 95% CI were calculated.Among 449 patients with type II diabetes mellitus, blindness was seen in 17 (3.79%) patients. Among them, 1 (5.88%) had severe nonproliferative diabetic retinopathy (NPDR), 3 (17.65%) had proliferative diabetic retinopathy (PDR) and 8 (47.06%) had severe diabetic macular oedema (DMO) .Blindness in either eye was found in 10 (58.82%) male and seven (41.18%) female. The mean age of the patients having blindness was 61.94\u00b115.24 years. Among 17 patients, 6 (35.29%) patients were of the age group 70 years or above whereas only 2 (11.76) patients were of the age group of 40 years or less. The mean duration of diabetes mellitus in the 17 patients was 14.37\u00b18.02 years. In only 7 (41.10%) patients, the duration of DM was 11 to 15 years whereas only 4 (23.53%) patients had DM duration of 21 years or more. No patients within 1 year or less duration of DM were blind. A total of 9 (52.94%) patients were only on oral hypoglycemic agents (OHA) and 8 (47.06%) patients were on both OHA and insulin .5In this study, the prevalence of blindness among the type II diabetes patients was 17 (3.79%) which is comparable to study conducted in Nigeria as both were hospital based study with similar study population.6 The prevalence of blindness in our study is lower than in Bhaktapur Retina Study where diabetic retinopathy was responsible for 5.74% of bilateral low vision and 7.89% of unilateral blindness in the patients and other studies done in Ghana, Nigeria and Jordan due to the different study criterias, settings and study population.10 Our prevalence was even more lower when compared to a study conducted in Northeast China where 29.6% of blindness was observed in patients with diabetic retinopathy while the leading cause of blindness was due to proliferative diabetic retinopathy in 45.4% of study cohort and another study conducted in Mexico had 29.1% of blindness.12 Their higher prevalence may be due to the fact that they included diabetic patients with cataract and uncorrected refractive errors also in their inclusion criteria which was exclusion criteria for our study population and maximum number of patients had short duration of diabetes in our study as it is known fact that more than 10 years of diabetes doubles the risk of visual impairment and blindness.13The prevalence of blindness varies widely and has been reported almost zero in most parts of Africa, to three to seven percent in South-East Asia and Western Pacific to high as 15-17% in America and Europe.16 Globally also, diabetic retinopathy accounts for five percent of all blindness and is the leading cause of blindness in people aged 15 to 64 years in industrialized countries which is higher than our study.2 Furthermore, our study was conducted among the known type 2 diabetics only and patients with undiagnosed diabetes might have lead to underestimation of visual impairment and blindness. But increasing awareness about DR and its ocular complications, improved management of diabetes and availability of laser and anti-VEGF treatment are likely to cause reduced prevalence of blindness in the future.Furthermore, studies done in England, Copenhagen and SN-DREAMS study in Chennai, India, the prevalence of blindness was lower than our study due to different study population and different settings.17Vision loss in approximately 25% of patients with DR is associated with progression of non proliferative DR to proliferative DR. PDR and DME are both sight-threatening conditions and can result in blindness. Worldwide, an estimated 17 million diabetic people have PDR and without appropriate treatment more than half of the patients with high-risk PDR will be blind within five years. In 1981 Nepal blindness survey by Nepal Netra Jyoti Sangh, 13.9% of blindness was due to diabetic retinopathy, posterior segment and Central nervous system diseases which increased to 17% in 2006 to 2010 blindness survey.18 Similarly, a study conducted in Tunisia also reported that prevalence of blindness was observed to be increased in severe DR where prevalence of blindness increased from 5.3% in mild and moderate NPDR to 18.7% in severe NPDR and progressed to 46.8% in PDR.13 In a Jordanian diabetic population also, blindness was significantly associated with severity of DR.10 Adequate literature are not available regarding the relation of blindness with severity of diabetic retinopathy in Nepal and could not be explored in this study too. However, primary interventions such as intensive glycemic and blood pressure control can reduce the prevalence of DR, while secondary interventions, such as laser photocoagulation, may prevent further progression of DR and vision loss. Although refractive error correction could improve useful vision significantly in patients with diabetic retinopathy, it cannot eliminate the visual loss.19In our study, the prevalence of blindness was observed to be higher in severe diabetic retinopathy. In our study, patients with mild NPDR had no blindness, in patients having blindness, 3 (17.65%) patients had PDR but 8 (47.06%) patients had severe DMO. About 50% of blindness is preventable by early detection and management of PDR and DME which can be done by regular follow up of patients with PDR and DME, laser photocoagulation and intensive blood sugar control.This study has few limitations. Data were collected from known diabetic cases only so patients with undiagnosed diabetes may have lead to underestimation of prevalence of blindness. This was a single hospital-based crosssectional study so results cannot be applied and interpreted to the general Nepalese population. Optical coherence tomography (OCT) was not used to evaluate macular edema in this study due to its unavailability so mild cases of DME may have been missed. Due to the cross-sectional study design, patients were not followed up so progression of blindness due to progression in DR may have been missed. However, this study provides the baseline data which may be useful for planning interventional strategies like primary prevention, prophylactic treatment with laser or intravitreal injections for management of PDR and DME. Nevertheless, such data estimates are very important for providing baseline data on the basis of which screening and public health programs can be planned to reduce the risk of blindness due to diabetic retinopathy.The prevalence of blindness among type II diabetic patients was lower than in other studies conducted in similar settings. Since our study population had a short duration of diabetes, we need to follow up these cases to assess for increasing severity of blindness so that appropriate interventions can be applied timely."} +{"text": "Life-history traits co-evolve and materialize through physiology and behavior. Accordingly, lifespan can be hypothesized as a potentially informative marker of life-history speed that subsumes the impact of diverse morphometric and behavioral traits. We examined associations between parental longevity and various anthropometric traits in a sample of 4,000\u201311,000 Estonian children in the middle of the 20th century. The offspring phenotype was used as a proxy measure of parental genotype, so that covariation between offspring traits and parental longevity (defined as belonging to the 90th percentile of lifespan) could be used to characterize the aggregation between longevity and anthropometric traits. We predicted that larger linear dimensions of offspring associate with increased parental longevity and that testosterone-dependent traits associate with reduced paternal longevity. Twelve of 16 offspring traits were associated with mothers' longevity, while three traits robustly predicted fathers' longevity. Contrary to predictions, mothers of children with small bodily dimensions lived longer, and paternal longevity was not linearly associated with their children's body size (or testosterone-related traits). Our study thus failed to find evidence that high somatic investment into brain and body growth clusters with a long lifespan across generations, and/or that such associations can be detected on the basis of inter-generational phenotypic correlations. According to the theory of life-history evolution, the key traits that contribute to Darwinian fitness, i.e., growth, survival and reproduction, evolve in a coordinated manner. The theory views the evolution of these traits as the product of interactions between intrinsic constraints and trade-offs as well as extrinsic factors in the environment that affect mortality risk and resource availability \u20133. TradeLife-history traits materialize through physiology and behavior. This means that in addition to basic components of fitness, such as fecundity and age-specific survival probability, values of anatomical, physiological, behavioral and psychosocial traits too accumulate non-randomly among the individuals within populations , 3, 6, 7Life-history (and related) traits co-evolve because they adapt to the same environment. For example, low levels of extrinsic and intrinsic mortality typically favor coevolution and genetic clustering of qualities characteristic to slow pace of life\u2014including slow maturation and high somatic investment into body and brain growth, delayed reproduction, long lifespan, conscientious personality and propensity for relatively high parenting effort in relation to mating effort , 12, 13 h2 = 0.25\u20130.4; reviewed by (rg = \u22120.40), age of first birth (rg = 0.33) and years of schooling (rg = 0.26) . With th = 0.26) . Psychom = 0.26) ]. A rece = 0.26) .Lifespan can thus be considered a potentially informative marker of life-history speed that subsumes the impact of many anthropometric traits. However, measuring phenotypic correlations between lifespan vs. other traits of interest at the level of individuals (though interesting on its own merit) does not enable to distinguish between phenotypic and microevolutionary trade-offs. In intergenerational studies, this problem can be indirectly circumvented by taking advantage of the fact that children inherit 50% of each of their parents' genomes so that the phenotype \u201326 or geSo far, few studies have used offspring morphometric traits to predict parental longevity and lifespan, and all of these were based on parent-son comparisons. In a study of an entire community of Tecumseh, Michigan, the longevity of either parent was related to high values of ventilatory lung function among the sons . A studyrg = 0.30), childhood height (rg = 0.12), adult hip circumference (rg = 0.28) and parental mortality. At the same time, birth weight was negatively associated with parental mortality (rg = \u22120.21) (Genome-wide association study in the UK Biobank (UKBB) that examined parental survival in relation to offspring genotype (pooled over sexes of offspring and parents) demonstrated positive genetic correlations between childhood obesity (= \u22120.21) . Another= \u22120.21) . Height-= \u22120.21) .Here we examine associations between parental longevity and 16 anthropometric traits of their children, measured at school age. The dataset collected by Prof. Juhan Aul in the middle of 20th century Estonia see , 35] is is 35)] As regards general predictions about the association between offspring phenotype and parental longevity, opposing scenarios can be imagined. On the one hand, smaller individuals can be predicted to live longer than larger ones because of decreased cancer risk due to a smaller number of cells whose replication involves the risk of DNA damage . In addiStudied traits include leg length . DeAge- and sex-specific residuals of anthropometric traits were calculated from generalized additive models in which the focal trait was regressed against smooth non-parametric functions of age (in days) and birth date using package \u201cgam\u201d for R and [R ]. Residun = 11,502) and/or mothers age of death was recorded in the Estonian Population Registry as at 2018. Sample sizes vary between analyses because participants differ with respect to the number of anthropometric and biosocial traits recorded. To eliminate reverse causation 20), compWe analyse the association between offspring phenotype and parental longevity/mortality among the sample of children pooled over sexes and also for sons and daughters separately because pedigree studies have shown that inheritance patterns of longevity/lifespan may depend on the sex of parents as well as their offspring [reviewed in ], likelyThe odds of becoming longevous . Models for fathers were adjusted for the three-level factor of SEP . For instance, the R syntax for the model testing whether paternal odds of becoming longevous depended on the height and its squared value [adjusting for fathers' year of birth and socioeconomic position was as follows: lrm]. Additionally, we run all the models with parental offspring number and its squared value to test for the possibility that the curvilinear association between offspring number and parental longevity would afData processing was performed anonymously under the license of the Research Ethics Committee of the University of Tartu and approved by the Estonian Data Protection Directorate .P = 0.040; The number of children was associated with the maternal probability of becoming longevous in a non-linear way, increasing with the number of children from one to six and decreasing with further increases in parity . Among tNon-linear associations between measures of offspring body size and mothers' longevity were detected for two traits. Mothers' odds of becoming longevous decreased with the leg length of their daughters in a non-linear, U-shaped manner in ESM.P = 0.008; The number of children was associated with paternal probability of becoming longevous in a non-linear way, increasing with the number of children from one to four and decreasing with further increases in parity . LongeviThree traits showed bell-shaped associations with fathers' odds of becoming longevous . FathersThe most prominent finding of this study is that many anthropometric traits of offspring were linearly associated with longevity of their mothers, while only three offspring traits predicted longevity of fathers in a straightforward way. One explanation might stem from the sex difference in lifespan in Estonia, which has been among the largest in Europe since at least 1980 . UnlikeSince from the mid-1990s, mortality rates from treatable causes (mainly diseases of the circulatory system and some curable cancers) have been decreasing in Estonia , it is pAnother, mutually not exclusive explanation for why offspring traits predicted longevity better in the case of mothers than fathers might relate to maternal effects. For instance, maternal hyperglycaemia during pregnancy has been associated with increased offspring adiposity in childhood . Under tA possible explanation of why mothers but not fathers of small-bodied children became longevous might be the sex-specific cost of reproduction, i.e., energetic and somatic costs incurred throughout pregnancy and lactation of large-bodied babies . Note, hIt may also be possible that children are more similar to their mothers than to fathers with respect to anthropometric traits or that the associations between mortality and own anthropometric traits are stronger among women than among men . InheritYet another explanation for the sex differences of associations between anthropometric traits of children and longevity of their parents might stem from different causes of death between the mothers and fathers of participants. During the study period, the three most common causes of death in Estonia were diseases of the circulatory system, neoplasms, and external causes, i.e., deaths due to injuries and poisoning. Diseases of the circulatory system were more common among women than men of offspring traits and parental survival beyond the age corresponding to the 90th/99th survival percentile , 20, 23 Our findings of associations between the height of children and parental longevity differ from those of the Swedish study of military conscripts, which showed that both mothers and fathers of taller sons had lower overall mortality . In our rg = 0.12) [ associations of height and leg length with all-cause (and particularly cardiovascular and respiratory disease) mortality found in a large number of studies in developed countries . Although height is often positively associated with many forms of non-smoking related cancers (= 0.12) [; see als= 0.12) . Altogerg = 0.30 is partly consistent with findings of the study performed in Michigan during 1959\u201360 and showing that greater longevity (death after age 65) of either parent was associated with high values of ventilatory lung function among the sons . InteresProbably the most parsimonious explanation for the observed patterns is that the associations between paternal longevity and the robustness of their sons mainly stem from genetic causes. Because children share the same socio-economic and lifestyle confounders with both of their parents, we should have seen similar associations between offspring traits and longevity of both mothers and fathers in case if such associations were mainly caused by external environmental influences such as socially inherited SEP. Further, the inverse association between paternal longevity and daughters' lung capacity suggests that genetic associations between offspring phenotype and parental longevity are sex-specific. Regarding the longevity of mothers, sex-specific association emerged with respect to the cranial volume of their children: mothers of daughters (but not sons) with large heads had lower chances of becoming longevous . Sex-speThis study showed that children's anthropometric traits predicted their parents' longevity better in the case of mothers than fathers. Mothers of small-bodied children and fathers of vigorous sons had higher chances of becoming longevous. Some of our findings were consistent with previous empirical and theoretical knowledge about processes affecting lifespan. Other findings were unexpected, such as inconsistent relationships between different testosterone-dependent traits and cranial volume of offspring vs. parental longevity. In particular, our findings failed to support the predictions that traits characteristic of to slow pace of life, such as high somatic investment into body and brain growth, cluster with long life.One of the limitations of this study is the absence of information on the causes of parental deaths. For instance, the hypothesis that anthropometric traits that reflect testosterone exposure (and/or amount in circulation) associate with risk-prone behavior predicts that such testosterone-dependent traits associate specifically with mortality due to external causes in men. The absence of information about the causes of death of fathers prevented explicit testing of this hypothesis. Another limitation is the absence of information about possible sex-specific heritabilities of anthropometric traits and lifespan in the studied population. For instance, the sex-specific association between offspring traits and parental longevity could be explained if the inheritance of lifespan and/or morphometric traits would appear higher in the maternal than paternal line.The central contribution of this study is that anthropometric traits of offspring were differently associated with mortality of their mothers and fathers and that some of these associations depended on the sex of offspring. This implies that future studies of longevity (including GWAS) would benefit from analyzing the associations between parental and offspring traits in a sex-specific manner. An important question arising from this study is whether or how much our findings are characteristic of a population with an extensive (9\u201310 years) sex difference in life expectancy. Future research would benefit from studies in societies with a smaller gender gap in lifespan to test our results' generalizability.https://www.muis.ee/en_GB/museaalview/3451136.The raw data presented in this article is available in The studies involving human participants were reviewed and approved by Data processing was performed anonymously under the license of the Research Ethics Committee of the University of Tartu and approved by the Estonian Data Protection Directorate . Written informed consent for participation was not provided by the participants' legal guardians/next of kin because: The study was performed between 1956 and 1969, when Estonia was occupied by Soviet Union and written consent was not required for this kind of research at that time.MV located the data of schoolchildren, built up, and complemented the database. MV and PH designed the study and wrote the manuscript. RM performed the statistical analyses. All authors contributed to the article and approved the submitted version."} +{"text": "The incidence rate of thyroid carcinoma (THCA) markedly increased in the recent few decades and has been likely over-diagnosed, especially papillary thyroid cancer (PTC) in women. However, the incidence of advanced-stage papillary thyroid cancer is also rising. According to earlier studies, tumors with identical pathology might have different clinical outcomes, which implies some variances in papillary thyroid cancer. Although the mortality of thyroid cancer has remained stable or declined, there is still an important problem in estimating whether it is benign or needs surgery for patients with papillary thyroid cancer.After obtaining data from The Cancer Genome Atlas (TCGA) Project-THCA database by R package TCGA bio links, 18 samples (11 at stage IV as high-risk group and 7 at stage I as low-risk group) were obtained using survival package and edgeR to ensure differential expression; ClusterProfiler package was used to carry on gene set enrichment analysis and searched the possible pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. STRING and Cytoscape were used to construct and modify the protein\u2013protein interaction (PPI) network to get hub genes of differentially expressed genes. Next, the pROC package was used to get the receiver operating characteristic (ROC) curves of hub genes\u2019 disease-free survival (DFS). Then, transcription factors (TFs) and miRNAs of key genes were predicted by ENCORI and AnimalTFDB. In the end, TF\u2013target genes\u2013miRNA regulatory network was also constructed by Cytoscape.Our research obtained the top 9 candidate genes from the whole network . According to the ROC results, TIMP1, LOX, CD276, IFNA1, TLR2, and POSTN were considered to play a more critical role in malignant papillary thyroid cancer or immature cancer of papillary thyroid cancer. Our analysis concludes that TIMP1, LOX, CD276, IFNA1, TLR2, and POSTN are identified as thyroid cancer biomarkers, which lead to the different clinical courses of a woman older than 55 years old with papillary thyroid cancer. Especially CD276, POSTN, and IFNA1 may be considered as new biomarkers associated with the prognosis of thyroid cancer.TIMP1, LOX, CD276, IFNA1, TLR2, and POSTN have different expressions in PTCs, which lead to the various clinical courses of a woman older than 55 years old with papillary thyroid cancer. Especially CD276, POSTN, and IFNA1 may be considered as new potential biomarkers associated with the prognosis of thyroid cancer. In addition, TF\u2013miRNA\u2013target gene regulatory network may help further reach for PTC. The incidence rate of thyroid carcinoma (THCA) markedly increased in the recent few decades and has Controversial issues in thyroid cancer management mainly focus on treatment options for different thyroid cancers (DTCs) . RecentlThis research performed many analyses through a wide-ranging bioinformatics study to classify the critical hub gene in thyroid cancer with a poor prognosis. A total of 994 genes upregulated in the high-risk group were obtained from TCGA-THCA data by edgeR , 11. TheThe Cancer Genome Atlas (TCGA) research network has collected many public clinical and molecular profiling of more than 10,000 tumor patients across 33 different tumor types. We obtained the clinical and transcriptome profiling of THCA from the TCGA-THCA database by TCGA-biolinks packages, which provide several useful functions to search, download, and prepare TCGA samples for data analysis , using tGene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to reveal the functional enrichment analysis of DEGs. BiomaRt packages that include over 800 different biological datasets spanning were used to convert gene ID from other types , 19. Cluhttps://string-db.org/), an online database, was used to establish the protein\u2013protein interaction (PPI) network of the candidate genes, which evaluates the interacting units with protein-coding gene loci (STRING (isoform) , and theisoform) . Next, tThe receiver operating characteristic (ROC) curve is a commonly used graphical summary to evaluate the predictive value of biomarkers . To verihttps://www.ncbi.nlm.nih.gov/), transcription factors were predicted by AnimalTFDB and selected by score >20 (https://rna.sysu.edu.cn/encori/index.php) was used to predict miRNAs that bind to hub genes and apply standards including CLIP-Data \u22653, pan-cancer \u22651, and miRNA, with the least intersections in three databases selected (After the reference sequence (RefSeq) of the key genes were obtained from National Center for Biotechnology Information (NCBI) RefSeq database was considered statistically significant. Then, a PPI network was made with Cytoscape. A total of 905 nodes and 4,941 edges were included in that PPI network. Additionally, the modules in the PPI network were analyzed by MCODE, and >8 was set as cutoff criteria with the default parameters Figure\u00a04pROC packages made the receiver operating characteristic (ROC) curve to explore the patient\u2019s prognosis with high-expression hub genes. According to the results of the ROC curve, TIMP1, LOX, CD276, IFNA1, TLR2, and POSTN as key genes are more relevant to THCA Figure\u00a05A total of 47 miRNAs and 44 transcription factors that could be bound to key genes are predicted. TF, miRNA, and target gene relations were revealed in the regulatory network Figure\u00a07In recent years, thyroid carcinoma has had a worldwide and dramatic incidence, especially in women. However, thyroid cancer treatment options are limited, and they mostly need to have surgery and supply of thyroxine for the whole of their later life. The high-intensity treatment for most tumor diameters over 1\u00a0cm, especially patients with differentiated thyroid cancer or papillary thyroid cancer, has also been controversial after PTC prognosis is more remarkable than that of other tumors. The fact that advanced thyroid cancer is found with the increase in cardinality estimate also promotes more research to distinguish the difference from PTC so that more suitable and precise therapeutics could be suggested to each of the patients.For the above target, it is critical to know the prognosis of carcinoma, especially for PTC, which has a high risk of incidence and is likely over-diagnosed. Many studies have utilized bioinformatics technology to uncover biomarkers in THCA or PTC. However, the majority of them compared carcinoma and normal samples. According to the 8th American Joint Committee on Cancer (AJCC) and other experts, there may be distinct influencing factors for varying clinical outcomes in patients with PTC who are over 55 years old \u201334. In tIn the present study, 994 upregulated genes were obtained from TCGA-THCA data using the edgeR package. GO was enriched in cell connection and extracellular matrix organization. KEGG pathway enrichment analysis of those DEGs could be linked to thyroid cancer growth and metastasis pathways such as proteoglycans in cancer, PI3K-Akt signaling pathway, and p53 signaling pathway. After using MCODE and Cytoscape network analysis tools of Cytoscape, nine hub genes with greater degrees were finally obtained , which were considerably overexpressed in PTC with a poor prognosis compared to those with a better prognosis. In comparison to the survival analysis of nine hub genes, TIMP1, LOX, CD276, IFNA1, TLR2, and POSTN as key genes are more relevant to THCA.Tissue inhibitor of metalloproteinase 1 (TIMP1) belongs to the TIMP gene family, and its encoded protein can promote cell proliferation. TIMP1 was found to be overexpressed in classic and follicular variants of PTC in different age and gender groups and in the BRAF-MUT group compared to that in BRAF-WT patients in previous research , 36. It The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.L-QY designed the analyses. C-CL and MU analyzed the data and wrote the manuscript. XL and S-KS collected the data, YW and FL prepared the figures, and BG and M-HZ revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China , Natural Science Foundation of Hunan Province (No. 2021JJ40842), and Key R&D Plan Hunan Province (2020SK2078).We want to thank our colleagues in the lab for their assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."} +{"text": "Major trauma often results in long-term disabilities. The aim of this study was to assess health-related quality of life, cognition, and return to work 1\u00a0year after major trauma from a trauma network perspective.n\u2009=\u2009536) were selected from trauma region Southwest Netherlands. Eligible patients (n\u2009=\u2009365) were sent questionnaires with the EQ-5D-5L and questions on cognition, level of education, comorbidities, and resumption of paid work 1\u00a0year after trauma.All major trauma patients in 2016 response rate was obtained. EQ-US and EQ-VAS scored a median (IQR) of 0.81 (0.62\u20130.89) and 70 (60\u201380), respectively. Limitations were prevalent in all health dimensions of the EQ-5D-5L; 90 (50%) responders reported problems with mobility, 36 (20%) responders reported problems with self-care, 108 (61%) responders reported problems during daily activities, 129 (73%) responders reported pain or discomfort, 70 (39%) responders reported problems with anxiety or depression, and 102 (61%) of the patients reported problems with cognition. Return to work rate was 68% . A median (IQR) EQ-US of 0.89 (0.82\u20131.00) and EQ-VAS of 80 (70\u201390) were scored for fully working responders; 0.77 and 70 for partial working respondents; and 0.49 and 55 for unemployed respondents.A 50% (The majority experience problems in all health domains of the EQ-5D-5L and cognition. Return to work status was associated with all health domains of the EQ-5D-5L and cognition. In the global burden of disease, trauma is a major contributor to death and disabilities . SurviviIn the past decades, trauma networks have been implemented. Such networks help to improve outcome of trauma care , 5. PrimOver the past 2 decades, many studies have been conducted on quality of life (QoL) after major trauma . These publications mainly focus on MT patients admitted to designated (major) Trauma Centers (TC). However, many MT patients also being admitted to non-Trauma Centers (NTC) . Only a Most trauma registries identify the severity of injuries with the Injury Severity Score (ISS) . which iEuroQoL 5D-3L is predominantly used as an instrument to measure QoL in the previous studies on MT. Since EQ-5D-5L was introduced, more differentiation is possible in distinguishing between minor levels of impairment. QoL studies focusing on MT that have used EQ-5D-5L in combination with injury coding using recent AIS revision are scarce .The aim of this study was to assess health-related quality of life, cognition, and return to work (RTW) 1\u00a0year after MT from a regional trauma network perspective.The local Medical Research Ethics Committee exempted this study. Following review of the protocol, they concluded that the study is not subject to the Medical Research Involving Human Subjects Act (MEC-2017-041).Trauma region Southwest Netherlands is a large and diverse trauma region, with a level I TC, 11 level II/III NTC hospitals, a Burn Center, a dedicated Eye Hospital, and three Emergency Medical Services (EMS). It consists of rural, remote, industrial, urban, and touristic areas with a dense infrastructure inhabited by 2.5 million people. It has an even larger catchment area with the availability of Helicopter Emergency Medical Services (HEMS).Trauma region Southwest Netherlands participates in the Dutch National Trauma Registry (DNTR) with theAll 12 DTR SW hospitals participated in this study. One year after trauma, the municipal base administration was checked whether or not patients had died. Each week, included patient injured 12\u00a0months prior were contacted. Non-fatally injured patients living in The Netherlands or the Flemish region of Belgium were sent a patient information letter, an informed consent form, and a questionnaire in Dutch. All questionnaires were self-reported, but respondents were allowed to ask for help. If patients lived in the Netherlands but had a language barrier, they were advised to complete the questionnaire with a relative that could help linguistically. For children younger than 13, it was compulsory to complete the questionnaire and consent together with their parents or legal guardian. Children between 13 and 18\u00a0years of age were obliged to include consent of a parent or other guardians and allowed to complete the questionnaire themselves. If adult patients were incapacitated, proxies were allowed to sign informed consent. Patients who did not respond after 1\u00a0month were contacted by telephone until contact. All questionnaires contained the adult version of the EQ-5D-5L.To determine whether or not the responders were a representative sample of the population, demographic, injury-related parameters, as well as the probability of survival, were compared between responders and non-responders.Comorbidities were surveyed with a modified version of the Cumulative Illness Rating Scale (CIRS), which is validated as an indicator of health status .Educational level was trichotomised in \u2018low\u2019 , \u2018middle\u2019 , and \u2018high\u2019 (university of applied science or academic).Health-related QoL was measured by the EQ-5D-5L which is a valid instrument for measuring QoL in healthy, chronically ill, and trauma populations \u201320. It cWorking age population was considered to be 18\u201365\u00a0years. Patients were asked how many days and how many hours per week they had paid work before and 1\u00a0year after trauma. RTW was trichotomised in \u2018full RTW\u2019, \u2018partial RTW\u2019, and \u2018no RTW\u2019. Subgroup analysis on the level of RTW was done for gender, age, type of injury, ISS, and MAIS. RTW was also analyzed in relation to each health domain of the EQ-5D-5L and cognition.Statistical analyses were done with Statistical Package for Social Sciences version 24.0 .Normality of continuous variables was tested using the Shapiro\u2013Wilk test. All continuous variables were non-normally distributed. Descriptive statistics are presented as median for continuous variables and number (percentage) for categorical variables.p value of 0.05 was considered significant.A Mann\u2013Whitney test was used for comparing two groups. A Kruskal\u2013Wallis test was used for multiple groups, and in case of significant differences, groups were tested pairwise with Mann\u2013Whitney tests. For nominal variables, a Chi-square test or Fisher\u2019s exact test was used as applicable (both two-sided). A n\u2009=\u2009139), double registries due to inter hospital referrals (n\u2009=\u200913), and residency abroad (n\u2009=\u200919). This resulted in a total of 369 patients who were sent questionnaires of whom 185 responded (50% response rate). The median follow-up time was 384\u00a0days (IQR 372\u2013414). The responding group was significantly older than the non-responding group with a median age difference of little over a decade . Overall, the responding grouped seemed representative for all included patients that were sent questionnaires were 0.81 (IQR 0.61\u20130.89) and 70 (IQR 60\u201380), respectively.Eighty (44%) responders reported to be worse off than before trauma. The median EQ-US and EQ-VAS of all respondents responders reported limitations with mobility, 36 (20%) responders reported limitations with self-care, 108 (61%) responders reported limitations during daily activities, 129 (73%) responders reported limitations due to pain or discomfort, and 70 (39%) responders reported limitations with anxiety or depression. In the Dutch reference population , percentCognitive limitations were reported by 102 (61%) responders. In the Dutch reference population , 7.5% ren\u2009=\u200921) and 18\u201355 (n\u2009=\u200984) was present for EQ-US . Age group 0\u201317 (n\u2009=\u200921) scored significantly higher (p\u2009<\u20090.001) than age groups 18\u201355 (n\u2009=\u200984) and 55\u2009+\u2009(n\u2009=\u200977) on their general health state . Having comorbidities was associated with a worse EQ-US , a worse EQ-VAS , and more cognitive limitations . Educational level was linear associated with cognitive limitation .Table n\u2009=\u200954) scored significantly lower than patients with ISS 16\u201324 (n\u2009=\u2009128) on EQ-US and EQ-VAS with a median utility sum score of 0.66 and 0.84, respectively (p\u2009<\u20090.001), and a median VAS of 60 and 75, respectively (p\u2009<\u20090.001). Patients with ISS\u2009\u2265\u200925 (n\u2009=\u200954) reported cognitive limitations significantly (p\u2009=\u20090.003) more often than patients with ISS 16\u201324 .Patients with an ISS\u2009\u2265\u200925 than patients with moderate (MAIS 1\u20132) lower extremity injuries or patients without lower extremity injuries . Patients with severe spine injuries (MAIS\u2009\u2265\u20093) scored significantly worse on EQ-US than patients without spine injuries . Patients with severe head injuries had significantly more often cognitive limitations than patients with moderate or no head injuries . Patients with moderate and severe face injuries reported significantly (p\u2009=\u20090.043 and p\u2009=\u20090.025 respectively) more often cognitive limitations than patients with no injuries in the face .Patients with a severe lower extremity injury (MAIS\u2009\u2265\u20093) had significantly lower overall scores (n\u2009=\u200968), 37% (n\u2009=\u200937) worked as much or more as before trauma, and 31% (n\u2009=\u200931) partially resumed work had a median (IQR) EQ-US and EQ-VAS of 0.89 (0.82\u20131.00) and 80 (70\u201390), respectively. This differed significantly with the partial working group , p\u2009<\u20090.001; EQ-VAS 70 (62\u201380), p\u2009=\u20090.001) and the unemployed group , p\u2009<\u20090.001; EQ-VAS 55 (40\u201372), p\u2009<\u20090.001). Partial and no RTW also differed significantly on the EQ-US (p\u2009<\u20090.001) and EQ-VAS (p\u2009=\u20090.002). Comparing RTW status per health domain gave significant results for all health domains and cognition. A better RTW status was associated with better scores on all health domains of the EQ-5D-5L and cognition. Patients who returned fully to work could still experience limitations in health domains (from 5% (n\u2009=\u20092) in self-care, up to 65% (n\u2009=\u200924) in pain or discomfort).All health domains including cognition are displayed per RTW group in Fig.\u00a0n\u2009=\u200968) returned to work more often (p\u2009=\u20090.020). Level of work resumption was unrelated to gender, type of injury, age, level of education, no. of comorbidities, and (severity) of specific organ injuries 1\u00a0year after MT.In Table This study assessed functional limitations by the means of health-related QoL and RTW in patients 1\u00a0year after MT (ISS\u2009>\u200915) from a trauma network perspective. The median overall utility score was 0.81 and the median general health state 70. Over 60% reported cognitive problems and less than 40% fully returned to work. If patients reported limitations, the majority scored little or moderate problems on all health domains. Severe problems or \u2018not able\u2019 was scored less (4.4\u201320.7%). Limitations within the five health domains of the EQ-5D or cognition were reported by 87% of all responders. In contrast, 44% of the respondents reported to be worse off after their trauma. Many respondents might have had functional limitations before their trauma. Trauma populations are associated with higher pre-existing morbidity, compared with non-injured populations, which might be prone to an overestimation of problems post-injury . In addiA mean overall utility score of 0.88 has been reported for the Dutch population . Dutch nDutch studies \u201329 compaA straightforward comparison of the present study with international publications on QoL after MT is difficult due to the use of different questionnaires, reporting the same instruments differently, variability in inclusion criteria, and the usage of different AIS revisions for injury coding. Studies that used unknown or older AIS revisions reported a mean EQ-US range of 0.60\u20130.69 or a median EQ-US range of 0.60\u20130.73, within a response time range of 6\u201318\u00a0months after trauma. The current study combined injury coding and inclusion on the basis of AIS08 with the EQ-5D-5L. Only one MT study with AISEspecially, international studies handling QoL after MT from a regional trauma network perspective are scarce , 9, 10. Patients with an ISS\u2009\u2265\u200925 scored worse on EQ-US, EQ-VAS, and cognition than patients with an ISS 16\u201324. A similar association was found between RTW and ISS. These results suggest that being able to return to work has a positive impact on the perceived QoL 1\u00a0year after MT, or that improved QoL may facilitate RTW. QoL and RTW are associated. Even though RTW can indicate a certain health status, the question is whether it is a reliable parameter to evaluate trauma outcome, since it can be affected by many other factors .First, a response rate of 50% might have resulted in a non-response bias. A part of the patients will not have been able to respond due to (severe) problems with, e.g., hand writing, post-traumatic stress, depression, or level of consciousness. MT patients that have had fewer problems post-injury might have been more likely to respond, because they have fewer attention consuming activities around their recovery or found participating not that confronting. These biases will result in an overestimation of the recovery status of the studied cohort 1\u00a0year after MT. However, during reminder calls, statements on not participating due to no experienced problems were also not uncommon. Recall bias could have resulted in an underestimation of the overall health state of the responders. In general, reliable information on pre-injury is lacking in this study. Such information is appropriate in compensating for attribution bias.Second, the EuroQOL-group advises a proxy version for ages 0\u20137, the EQ-5D-Y for ages 8\u201311, and for ages 12\u201318, the adult version can be used . These versions are developed on the basis of the EQ-5D-3L, and at present, a utility value set for the EQ-5D-Y is lacking. In the present study, the adult version of the EQ-5D-5L was used for children, which probably resulted in an overestimation of the QOL of children aged 15\u00a0years and younger .Third, the definition of MT has always been under the debate, we used a threshold of ISS\u2009>\u200915. The ISS was calculated following AIS08, while the most recent version being AIS15. It is unclear what the effects of the AIS15 are on an MT cohort with an ISS\u2009>\u200915. In addition, a certain part of injuries are wrongly coded due to inter-rater agreement and reliability limitations of the AIS . Adding The extent of problems people experience 1\u00a0year after MT stresses the necessity of a long multicenter follow-up. More insight can be gained in the long-term recovery status of MT patients. Physical and mental recovery take a long time, but social integration might take much longer; especially when not just considering the effect of traumatic events on individuals, but also on their social environment and relatives.One year after major trauma, the majority of patients experience problems in all health domains of the EQ-5D-5L and cognition, and function below population norms. Trauma care should focus on these health domains in collaboration with other revalidation disciplines. Return to work status was associated with all health domains of the EQ-5D-5L and cognition, showing that focus on health status of trauma patients in recovery trajectories can potentially have many positive effects."} +{"text": "MDR1 gene belongs to the ATP binding cassette family; it is known as one of the chemotherapy-resistant causes of AML. We aimed to study FLT-3ITD mutations and their association with MDR1 gene expression in AML individuals. Acute myeloid leukemia (AML) is a hematopoietic malignancy caused by genetic abnormalities. Currently, molecular and genetic factors are routinely used as diagnostic and prognostic markers. FLT-3 is one of the most known diagnostic factors in AML. FLT3-ITD mutation was assessed by polymerase chain reaction (PCR); Real-time quantitative PCR was performed to measure the amount of MDR1 gene expression. Bone marrow and blood smears of patients were evaluated in terms of morphology. SPSS 16.0 was used for data analysis.For investigation, 80 AML individuals and 20 healthy controls were selected. This study was done in the Cancer molecular Pathology Research Center of Mashhad University of Medical Sciences (MUMS), Iran during 2017-2019. FLT3-ITD mutation and MDR1 overexpression were found in 18.8% and 23.8% of AML patients, respectively. Statistical analysis did not show any relationship or association between these two markers. Cuplike morphology was observed in blast cells in 21.25% of AML cases, which was associated with the presence of FLT3-ITD mutation. FLT-3 and MDR1 function independently. Survival studies to determine the exact role of MDR1 overexpression in drug resistance issues would be suggested. Acute myeloid leukemia (AML) is an aggressive and proliferative disorder involving hematopoietic cells , 2. It cFLT3 mutations, including ITD and TKD subtypes, are among the most effective and prevalent genetic alterations. ITD type is more prevalent, and TKD is rare. FLT3-ITD mutations are detectable in 33% of AMLs, and mutation of the tyrosine kinase domain (TKD) occurs in ~ 10% of AML patients; FLT3 mutations are associated with decreased overall survival and higher rates of relapse and are more common in normal karyotype ones abnormality is reported in many cancers . It belosurvival .MDR1 gene expression and the presence of FLT3 mutation; the present study was designed due to a lack of sufficient information in the cited area. We searched many studies in the molecular field of AML, but limited papers have evaluated the relationships between the amount of PatientsThis case-control study was performed in the Cancer Molecular Pathology Research Center and hematology lab of Ghaem Hospital from October 2017 to March 2019. In this survey, 80 AML patients, including 44 men and 36 women, from different subtypes were examined; twenty healthy individuals were considered as a control group. Cases with secondary myelodysplastic syndrome (MDS), secondary AML, and Down syndrome were excluded from the patient group. AML was diagnosed according to the French-American-British (FAB) classification, Immuno-phenotypic characteristics, and laboratory findings; the AML subtype was also classified according to FAB classification.Bone marrow and peripheral blood specimens were collected in EDTA container tubes. Patients' peripheral blood and bone marrow smears were carefully evaluated for cell morphology. Blast and differential counts were performed, and the blasts' morphology and other cells were considered.RNA ExtractionTotal RNA was extracted from mononuclear cells of the samples using the TriPure Isolation Reagent Kit ; nanodrop was used for evaluating the extracted RNA. Patient RNA samples were loaded in the agarose gel with loading dye; approximately the 28S rRNA band should be twice as intense as the 18S rRNA. Extracted RNA was stored at -80 \u00b0C until the test time.c-DNA Synthesis Complementary DNA (cDNA) synthesis was done by Revert Aid TM cDNA Synthesis Kit ; cDNA was used as a template for PCR amplification for FLT3/ITD mutation identification and MDR1 gene expression. Analysis of the Flt3/ITD2, dNTPs, Taq DNA polymerase , cDNA (1:50). Exons 11 and 12 of the FLT3 were ampli\ufb01ed by polymerase chain reaction (PCR) using the following primers: 5'TGGTGTTTGTCTCCTCTTCATTGT-3` as the forward and 5`-GTTGCGTTCATCTTTCAAA-3` as the reverse one. The PCR was done in 10x PCR buffer II, MgClReaction conditions, including denaturing, annealing, and extension steps, were performed at 94\u00b0C for 30 seconds, 61\u00b0C for 30 seconds, and 72\u00b0C for 30 seconds for 35 cycles in the Thermal Cycler (Applied Biosystems). Also, the initial denaturation step was 5-minute at 95\u00b0C, and the \ufb01nal extension was 72\u00b0C for 5 minutes. Products were electrophoresed in 8% polyacrylamide gel and 4%\u00a0agarose\u00a0gel. The emergence of a single band in the 245 bp size indicated no mutation (wild type); a double band indicated a mutation in the FLT3 gene of the ITD type .Real-time Polymerase Chain Reaction (RQ-PCR)The real-time qPCR technique with the SYBR Green method was performed to evaluate the MDR1 mRNA expression in the AML and control samples. cDNA was used for this procedure. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was considered the reference one; it was applied for data normalization to correct RNA quality and quantity variations. This procedure was performed by SYBR Premix Ex Taq TM II kit on an ABI thermos cycler . Also, a 5-fold serial dilution of GAPDH c-DNA and MDR1 c-DNA was performed to determine the efficiency before the procedure. -\u0394\u0394CT method was performed for relative gene expression calculation. The specificity of products was confirmed by the melt curve.All samples were performed in Triplicate. The reaction mixture contains 4 \u03bcL of cDNA template, 0.4 \u03bcL of each primer in MDR1 gene in the control group was considered a cut-off value (1.5). Samples with the MDR1 expression higher than 1.5 were categorized as overexpressed cases (MDR1 positive), and MDR1 expressions less than 1.5 were known as negative.The mean expression of the Statistical AnalysisStatistical analysis was conducted using the statistical program SPSS software, version 16 . The association between the levels of MDR1 m-RNA expression with FLT3-ITD mutations was determined by Fisher's exact test. The association was considered statistically significant when P-value \u2264 0.05 was achieved.Patients Information FLT3-ITD was detected in 15 out of 80 (18.8%); FLT3-ITD frequency was equal in both groups of patients, including recurrent mutation and non-recurrent ones. FLT3 ITD mutation was more frequent in M3, M4, and M5 subtypes of AML. Molecular data of patients have been summarized in 3/\u03bcL, platelet count: 58.39/dl\u00b147.9 \u00d7103/\u03bcL, and blast: 63.28\u00b123%. The blast cell count in our patients with FLT3-ITD gene mutation was higher .The samples belonged to the archive of the Cancer Molecular Pathology Research Center of Mashhad University of Medical Sciences, Iran. Of the studied individuals, 55% (44) were male, and 45% (36) were female. The age range was 2-60 years; the mean age of studied patients was 38 years . FLT3-ITMDR1 gene expression level in the control group was 1.5\u00b10.56 and in AML patients was 4.64\u00b114.41, ranging between 0.003 and 61.141. The average MDR1 gene expression level was not statistically significant between FLT3-ITD positive patients and group of patients without FLT3-ITD mutation .The mean MDR1 overexpression was found in 23.8% of AML cases; overexpression was observed in 33% of M0, 33% of M1, and 25% of M4/5 subtypes. Besides, MDR1 gene overexpression was found in 26.5% of AML individuals without recurrent mutations and 19.3% of cases with recurrent mutations. This finding was not statistically significant (P=0.5). Evaluation of AML patients with recurrent mutations showed that the number of expressed gene was lower in cases with good prognosis mutations such as t , t , inv (P=0.339). 17), inv in compaFLT3-ITD mutation and MDR1 gene expression (P=0.499) by the exact fisher's exact test; in fact, they are not involved in their pathogenesis.No direct or reverse correlation was found between FLT3-ITD mutation was observed in 11 patients with cuplike morphology (11 of 17 patients (64.7%)). A strong correlation was found between FLT3-ITD mutation and cuplike morphology. This morphology was also detected in rare negative cases of FLT3 mutation (1-5%). Cuplike morphology is more possibly seen in peripheral blood blasts. Among 17 cases with this morphology, cuplike blasts were detected in the peripheral blood of 9 cases and were not observed in the bone marrow; so, to diagnose this morphology, a peripheral blood smear is preferable to bone marrow aspiration . piration .FLT3-ITD mutation and MDR1 gene expression in AML cases. Previous studies about the relationship between these two factors are limited. The results of the present study showed no statistical relationship between these two prognostic markers; actually, they function independently (P=0.499).In this study, we evaluated two prognostic factors including FLT3 mutations are prevalent among AML patients; approximately, they are found in 25 -35% of AML cases. FLT3-ITD is the most common type among FLT3 mutations family, which is detectable in 20-27% of adults and 10-12% of children (10-14). ITD type frequency has been reported by Yuslina Mat Yusoff in Malaysia and Bhatldren 10-. ITD typ studies -19. Rega studies . FLT3-ITD and MDR1 genes are two independent factors, and none of them affect the others. Nasilowska et al. studied the FLT3-ITD influence on MDR1, MRP1 and BCRP mRNA expression, which are related to AML progression. They reported higher expression of MDR-1 in patients who were negative for FLT3-ITD; they believed that more investigations are required to find any correlations .Many studies evaluated the correlation between the FLT3-ITD mutation and cuplike morphology (fish mouth blast) are associated with each other; this finding has been reported by other studies, too .Contributor 1, 2 designed the research. Contributor 1, 2, 4, 5, 7 carried out the experiments. Contributor 3, 6 wrote the manuscript with support from Contributor 1, 2. Contributor 3, 8 performed the statistical analysis of the date. Contributor 1, 4 worked out almost all the technical details and performed the calculations for the suggested experiments. All authors participated in discussion of the results and made comments and suggestion on the manuscript and contributed to the research. The authors declared no conflict of interest.Mashhad University of Medical Sciences, Mashhad, Iran.This study was fully sponsored by"} +{"text": "Osteoporosis (OP) and Dermatoporosis (DP) are expressions of the aging process at the skin and bone levels, respectively. Both conditions are associated with increased morbidity for elderly people, and this requires necessary interventions. They share many common risk factors; among these, vitamin D (VD) deficiency appears to have a role. VD is involved in either disease with many mechanisms, among which immunomodulation. VD deficiency has been linked to OP because it inhibits the body\u2019s capacity to absorb calcium and maintain optimal bone health. Available evidence suggests that proper vitaminosis D also appears to be vital in preventing skin age-related issues. DP is often seen in elderly individuals, particularly those with long-term sun exposure and a history of chronic sun damage. VD deficiency can be linked to DP, since its involvement in collagen production, epidermal barrier function, inflammation regulation, wound healing, and sun protection. Aim of this review is to summarize the most updated existing evidence on the role of VD in the development of fragility syndromes such as DP and OP and the possible benefits of VD supplementation as a simple and harmful weapon against aging. It is distinguished by low bone mineral density (BMD) and decreased bone strength, which increases the risk of fragility fractures. OP is the leading cause of bone fractures in the elderly, making it a substantial public health issue with a large impact on health systems . Kaya an22.1OP is a systemic skeletal illness marked by low bone mass, micro-architectural degeneration of bone tissue, bone fragility, and an increase in fracture risk due to even minimal trauma . The epi2.2Under physiological conditions, skeletal homeostasis is guaranteed by an appropriate ratio between formation and resorption of bone tissue. Skeletal homeostasis is maintained under physiological settings by a balance of bone production and bone resorption. This adjustment is altered in pathological circumstances in favor of osteoclast-mediated bone resorption .This bone regeneration cycle decoupling is exactly what happens in elderly people: the decreased osteoblast activity determine a longer time required to fill resorption cavities and there is a low-grade systemic inflammation, especially involving pro-inflammatory cytokines which determine an increase in the amount and functioning of osteoclasts. As a result, in older persons there is an overall decline in bone with time. A negative calcium balance resulting from decreased dietary intake, reduced absorption and the compromise of kidney function, reduces the activation of VD and the calcium absorption from the gut . Osteocl2.3OP is asymptomatic for the majority of its clinical history; approximately one-third of fracture occurrences are silent, while the remainder present in pain. Osteoporotic fractures are fragility fractures, meaning they occur spontaneously or as a result of little trauma. In order of frequency, the most commonly affected sites are vertebra, femur, major non-vertebral/non-femoral fractures , minor fractures followed by pain. Vertebral compression fractures (VCFs) are the hallmark clinical presentation of OP. Unlike major posttraumatic VCFs, which are invariably symptomatic, those caused by moderate trauma are frequently misunderstood and thus go undiagnosed. They are typically asymptomatic or present with symptoms such as back or low back pain that responds to analgesic therapy , 16. The33.1Skin aging is not just an aesthetic problem. In fact, its effects also weaken the skin on a functional level, reducing the crucial protective properties of the skin. Since this growing knowledge in recent years, it has been necessary to coniate a term as DP that could focus the attention of clinicians on the urgency of preventing and treating this condition as well as OP.DP is a clinical entity that includes the whole broad spectrum of skin alterations due to aging, including complications that can cause severe morbidity for the affected elderly person, taking the form of a real chronic skin frailty syndrome . People Data on the prevalence of the disease are currently scarce, however they show the high frequency among the elderly and especially in women. The frequency of dermatoporosis is 32%, based on a study of 202 hospitalized participants aged 60 to 80 years. DP was found in approximately 22% of females and 38% of males . Another3.2There are many factors implicated in the development of DP. First of all, the decline with aging in dermal hyaluronic acid (HA). In the elderly there is a thinning of the skin, which loses its resistance to mechanical forces. The firmness of \u201chealthy\u201d skin is provided by the ECM, whose major constituent is HA, a non-sulfated glycosaminoglycan that is mostly produced by fibroblasts. HA is a very hydrophilic material that decreases friction between collagen fibers and provides shear force resistance. Yet, as people age, fibroblasts lose their ability to make HA; as a result, the ECM loses volume and consequently is protective mechanical function, determining skin laceration from minimal trauma . The lac3.3Clinical manifestation is variable and includes morphological and functional alteration of the skin. Skin atrophy, senile purpura, and stellate pseudoscars are morphological signs of skin fragility. Sun-exposed areas of skin atrophy include the pretibial zones, the back of the forearms, the dorsum of the hands, the presternal area, and the scalp. Dermatoporotic skin is clinically very thin and transparent, with many wrinkles, senile purpura, and pseudoscars as compared to younger skin. There is a significant reduction in skin thickness, demonstrated by ultrasound . The dermis, which contains the majority of the subcutaneous fat, shows substantial elastosis, while the epidermis displays linearization with lack of rete ridges . Senile Functional manifestations of DP are skin lacerations, delayed wound healing, and subcutaneous bleeding with the development of deep dissecting hematomas (DHH) and in the most serious cases even large areas of necrosis . DHH is 4OP and DP share many common risk factors Figure\u00a0155.1Besides being a lipid-soluble vitamin, VD is a steroid hormone. Humans obtain VD from either sunlight exposure to UVB rays or minimally from the introduction with plant or animal foods, dietary foods and supplements, as VD2 or VD3 . VD is pBoth innate and acquired immunity are affected by 1,25(OH)2D3. VD and its metabolites influence innate immunity by promoting macrophage development and activation, which results in the production of defensins including cathelicidin and 2-defensin . Mice on5.2The interpretation of serum 25(OH)D has to take into account many factors, as levels can vary widely in different life periods, based on degree of exposure to sunshine , phototype, and nutritional status . There i5.35.3.1VD is required to increase the active intestinal absorption of calcium by 30-80% which, later, becomes available for multiple physiological processes and for the mineralization of the skeleton. Additionally, it promotes calcium reabsorption in the kidney\u2019s distal tubule. By encouraging osteoblast development and regulation as well as the generation of proteins including collagen, alkaline phosphatase, osteocalcin, and RANKL, 1.25 (OH)2D3 also has direct effects on bone. It controls both bone formation and resorption . Clinica5.3.2post-hoc analysis confirmed the usefulness in terms of increasing BMD of the VD supplementation only in the group with a baseline level of 25(OH)D \u2264 30 nmol/L D level lower than 30 ng/mL) is considered a risk factor of OP because of the mechanisms that increase resorption seen above . Epidemi0 nmol/L . Benefit0 nmol/L . In the 0 nmol/L . In any 0 nmol/L , 92. Thi0 nmol/L . As disc0 nmol/L ; it is p0 nmol/L , that it0 nmol/L .5.45.4.1Skin and VD have a special relationship: skin is in fact the only organ that can produce VD and its metabolites, also being at the same time a major target for this hormone as well . KeratinAs previously reported, the presence of VDRs in almost all immune cells suggests that they are one of vitamin D\u2019s primary targets, and various immunological indicators are controlled by VDRs action . This haOverall, VD has an immunomodulatory effect on T cells . VD inhi5.4.2Several studies on VD receptor mutant mice have put the basis for the knowledge of VD relevance in controlling aging in skin and many other tissues, as these mice developed typical phenotypic traits of premature aging such as skin and overall body atrophy as well as OP. By normalizing mineral VD, these phenotypic traits can be reversed , 120\u2013122Aside from its potential application in the treatment of skin aging for aesthetic purposes, there is evidence that VD can also play a role in the prevention and management of DP and its severe repercussions. Here we summarize some key ways in which VD influences skin health and its potential impact on DP.First of all, in DP there is an impairment of the collagen component of the skin . As discImpaired wound healing is a common characteristic of dermatoporotic skin . VD has Finally, VD is involved in protection against harmful UV radiation. DP skin is more vulnerable to sun damage and should be shielded from excessive sun exposure . The ideOnly one MR trial has looked at vitamin D status and skin phenotype. Higher observed serum 25OHD concentrations were linked to perceived age, skin wrinkling, and pigmented spots, according to research by Noordam et\u00a0al. on facial skin aging features in about 4500 Dutch individuals. However, according to genetic predictions, serum 25OHD was not linked to these skin characteristics. This seems to suggest that the cause of skin aging is exposure to UV-B light rather than serum 25OHD concentrations .5.5Everyone in the aging population, regardless of bone health status, should get enough VD . The Bon6Aging is marked by the continuous and progressive decline in organic functions and the increase of prevalence of chronic degenerative disease. OP and DP are expressions of this process at the bone and skin levels, respectively. Both conditions are associated with increased morbidity for elderly people, and this makes preventive interventions necessary. A first conclusion of our study is that since DP is a frequently observed condition by dermatologists, its presence might serve as a straightforward clinical indicator of bone frailty, encouraging healthcare providers to recognize and treat underlying OP. Furthermore, the two conditions share many risk factors, some not always editable such as corticosteroid use, others on which it is possible to intervene as VD deficiency. VD is involved in either disease with many mechanisms, among which immunomodulation Figure\u00a02More research with rigorous and reproducible evaluation is required to better understand the role of VD in the development of fragility syndromes as DP and OP, but since now it is advisable to maintain adequate levels of VD to prevent these conditions, as VD deficiency is a simply avoidable and curable condition with major health effects.FR and CDS contributed to ideation, drafting, and revising of the manuscript. FR, DS, CMT, AS, DAC, RA, ML and CDS, contributed to the literature search and drafting of the manuscript. AC contributed to ideation and revising of the manuscript. All authors have revised and accepted the final version of the manuscript."} +{"text": "Background and objectiveIntramedullary nailing can be considered the current gold standard for the treatment of diaphyseal tibial fractures. Nailing ensures good fracture stability, protection against malalignment, and quick mobilization. The suprapatellar (SP) approach of tibial nailing in the semi-extended position has recently been recommended as a safe and effective surgical technique; it has been gaining significant attention in the orthopedic literature, with fewer complications and reoperations. The approach has been shown to facilitate a reduction in fractures around the knee joint\u00a0in the semi-extended position, and the extended position of the lower leg allows for easier fluoroscopic imaging. In this study, we aimed to compare the outcomes between SP and infrapatellar (IP) approaches of intramedullary nailing\u00a0in patients with extra-articular tibial fractures.MethodA randomized control trial was conducted over a period of 1.5 years at our tertiary care hospital after obtaining approval from its institutional ethics committee. A total of 60 patients with extra-articular tibial fractures were included in the study, with 30 patients each in the SP nailing group\u00a0and the IP nailing group, based on randomized sampling and with the help of radiological exposure in SP and IP nailing as per a previous study. The groups were then compared in terms of KUJALA patellofemoral knee score, operative time, radiation exposure, and time of union.ResultsWhen comparing both groups, those treated with the SP approach had better outcomes, including reduced radiation exposure, less pain, decreased operative time, better KUJALA patellofemoral knee score, and faster union.\u00a0ConclusionBased on our findings of the comparison between SP and IP nailing approaches of extra-articular tibial fractures,\u00a0the former leads to better and safer outcomes than the latter. Tibial fractures are the most common long-bone fractures, with the highest prevalence in men between the ages of 15 and 19 years, and in women between the ages of 85 and 95 years. The average age of a patient sustaining a tibial fracture is 37 years, with\u00a0an average of 31 years in men and 54 years in women. Tibial fractures are caused by direct injuries, including high-energy bending, low-energy bending, and penetrating wounds, as well as indirect injuries, including torsional mechanisms and stress fractures [The tibia is often involved in open fractures due to its subcutaneous location. Severe open fractures are associated with a high rate of complications and poor long-term outcomes. In comparison to other types of fractures, tibial fractures have higher rates of malunion and non-union. Approximately 80% of tibial fractures are associated with fibula fractures .The infrapatellar (IP) approach is a widely accepted treatment modality for extra-articular tibial fractures, and it is characterized by the effective management of both simple and segmental fractures of the tibia. The IP approach has several advantages, such as a shorter operative time, faster union, and early mobilization . AnotherThe present study was conducted from December 1, 2020, to October 31, 2022, at a tertiary care hospital. The study population included all patients with extra-articular tibial fractures admitted to the orthopedics ward and who fulfilled the inclusion criteria. In light of the ongoing coronavirus disease 2019 (COVID-19) pandemic, the convenient sampling technique was used, as it was considered the most suitable method for us. Patients who met the inclusion criteria were asked to participate in the study, which comprised\u00a0all extra articular-tibia fractures (closed fractures and up to compound grade 2 fractures) and patiThe patients were divided into two groups - the SP group and the IP group - and\u00a0operated on by experienced surgeons at the institution. Each patient was taken to the operation theater and placed on a table. Spinal anesthesia was given, and a high thigh tourniquet was applied. Cleaning, painting, and draping were\u00a0all done by ensuring\u00a0necessary aseptic precautions. Limb exsanguination was done with the use of an Esmarch bandage for one minute, and the tourniquet was inflated to a pressure of 150 mmHg above systolic pressure.For the IP group, the anteromedial aspect of tibial tuberosity was palpated and marked, and a skin incision 3-4 cm in length was taken. The knee was taken in complete flexion, and the patella tendon was palpated. The patellar tendon was split vertically in the\u00a0center. An incision was made through the patella\u00a0tendon, which ensured an accurate entrance into the bone. Then, a curved awl was used to make an entry point just above and medial to the tibial tuberosity. From the entry made with the awl, a guide wire was passed with a slight bend of the tip. Upon entry into the canal, a typical grating sensation was felt. The fracture fragments were aligned manually for smooth passing of the wire. A nail, whose length was determined preoperatively, was then attached with a threaded bolt to the proximal zig and kept aside.\u00a0Serial reaming of the canal was done. The nail was inserted over the guide wire and further into the medullary canal in a rotating motion by applying pressure. For the placement of the distal screw, distal locking was done under fluoroscopy guidance using a free-hand 4.5-mm bolt of appropriate length. For the placement of the proximal screw, proximal locking was done through a proximal zig using sleeves through a stab incision over the medial side. The screw length was measured with a depth gauge, and a 4.5-mm bolt was placed and checked under fluoroscopy.For the SP group, the patient was placed in the supine position on the operating table, and the affected leg was placed with a bolster under the knee joint, thereby flexing it to 25-30 degrees. The C-arm was placed on the contralateral side. A 2- to 3-cm vertical skin incision was made above the base of the patella. The quadriceps tendon was bluntly dissected, and a vertical central split was performed in the tendon. An entry tube was then inserted, as shown in Figures The entry point on the anteroposterior view was located 10 mm in the lateral direction from the center of the tibial plateau and lateral to the tibial tubercle, as seen in Figure On the lateral view, the entry point was anterior to the anterior articular margin, as shown in Figure The guide wire was then inserted into the medullary canal, as seen in Figures The protection sleeve was then inserted. Through the sleeve over the guide wire, the medullary canal was opened to a depth of 4-6 cm in the proximal tibia with a short reamer. A ball-tip guide was inserted into the medullary canal and advanced past the fracture level and down to the distal tibia. Fluoroscopic imaging was used in both planes to verify that the wire was within the medullary canal, as seen in Figures For nail insertion, the length of the nail was determined by using\u00a0a proper measuring guide, as seen in\u00a0Figures The reaming was performed through the protection sleeve. Depending on the length of the tibia,\u00a0the reamer used at the SP entry was longer than that at the IP entry. Typical reaming was then done to a diameter that was 1-1.5 mm larger than that of the nail. The inner part of the sleeve protector was removed before the nail was inserted.For the placement of the distal screws, distal locking was done under fluoroscopy guidance using a free-hand 4.5-mm bolt of appropriate length. The placement of the proximal screws was done through a proximal zig using the sleeves through a stab incision over the medial side and anteromedial side. The screw length was measured with the depth gauge, and a 4.5-mm bolt\u00a0was inserted into the nail.Fluoroscopy was used in both views to verify that the nail was not protruding into the knee. The knee joint was given a thorough saline wash to clear the joint space from any kind of debris. A preoperative radiograph was then taken Figure .The patients were followed up postoperatively\u00a0with radiography\u00a0at 0, one, and six months, as seen in Figures A radiological assessment was done at four weeks postoperatively, and evidence of callous formation was seen. The final outcomes were measured with respect to time of union, operative time, complications, radiological exposure, and functional outcomes assessed using the KUJALA patellofemoral score. These parameters were measured for a duration of six months, and the results were analyzed\u00a0by using appropriate statistical tests.The\u00a0study was approved by the institutional review board of the N. K. P. Salve Institute of Medical Sciences & Research Centre and Lata Mangeshkar Hospital, Digdoh Hills, Hingna Road, Nagpur .Table Table Table 2)\u00a0between the groups. An\u00a0unpaired Student\u2019s\u00a0t\u00ad\u00ad-test\u00a0showed a\u00a0highly statistically significant difference\u00a0between the groups (p=0.0001), with higher radiological exposure in\u00a0the\u00a0IP\u00a0group.Table Table Table Tibial shaft fractures account for 3% of all fractures in adults, and the conventional treatment for the condition involves intramedullary interlock nailing. Until recently, the IP approach was the most commonly used method of nailing, but the SP approach, with the knee in a semi-extended position, has gained popularity because of its many advantages, such as easy reduction, accurate entry, short operative time, and low exposure to fluoroscopic radiation .There have been concerns of malalignment with regard to the IP approach, with issues including knee hyperflexion, and difficulty in achieving and holding reduction in upper-third and lower-third fractures in hyperflexion. Various studies have reported a significantly lower incidence of malalignment when using the SP approach. While it is usually employed in proximal tibia fractures, better outcomes have also been seen in lower-third fractures. The SP approach does not require hyperflexion and repeated manipulation for positioning, which are prerequisites in IP nailing. Thus, the leg remains in a semi-extended position during the entire operative duration .The present study was conducted at our tertiary care institute with a view to comparing the functional outcomes of intramedullary interlock nailing between SP and IP approaches in patients with extra-articular tibial fractures. We included a total of 60 cases in our study, of which 30 were in the IP group, and 30 were in the SP group.In our study, the Student\u2019s t-test showed a significant difference between the mean duration for the union of fracture in both groups (p=0.0001), with a longer mean union time in the IP\u00a0group. Metcalf et al. have obsIn our study, the mean operative time was 147.33 \u00b1 8.96 minutes\u00a0in the IP group and 110.20 \u00b1 5.79 minutes in the SP group. Student\u2019s unpaired\u00a0t-test showed a highly statistically significant difference between the groups (p=0.0001), with a longer operative time in the IP group. Similar findings were seen in a study by\u00a0Al-Azzawi et al. [2\u00a0in the IP group and 38 \u00b1 30.6 cGY/cm2\u00a0in the SP group (p<0.05). In contrast, studies by Cicekli et al. [In the present study, Student\u2019s unpaired\u00a0t-test showed a highly statistically significant difference in mean radiological exposure between the groups (p=0.0001), with higher radiological exposure in the IP group. This is in line with the findings of Al-Azzawi et al. , where ti et al. did not Although an overall improvement in the KUJALA score was seen in both the IP and SP groups, the SP group saw much greater improvement. Student\u2019s unpaired\u00a0t-test showed a significant difference in postoperative KUJALA scores at one month and six months between the\u00a0groups (p=0.0001), with higher scores in the SP group. Similar findings were seen in a study by\u00a0Jones et al. , with\u00a0hiEastman et al. reportedLimitations of the studyThis study has one limitation that needs to be taken into account:\u00a0abrasions or wounds over the SP area that made it difficult to make an incision for SP nailing.Based on our findings in comparing the outcomes between SP and IP nailing of extra-articular tibial fractures, we found that the former was a better and safer method than the latter."} +{"text": "Cardiovascular diseases are the leading cause of death in industrialized nations. Due to the high number of patients and expensive treatments, according to the Federal Statistical Office (2017) in Germany, cardiovascular diseases account for around 15% of total health costs. Advanced coronary artery disease is mainly the result of chronic disorders such as high blood pressure, diabetes, and dyslipidemia. In the modern obesogenic environment, many people are at greater risk of being overweight or obese. The hemodynamic load on the heart is influenced by extreme obesity, which often leads to myocardial infarction (MI), cardiac arrhythmias, and heart failure. In addition, obesity leads to a chronic inflammatory state and negatively affects the wound-healing process. It has been known for many years that lifestyle interventions such as exercise, healthy nutrition, and smoking cessation drastically reduce cardiovascular risk and have a preventive effect against disorders in the healing process. However, little is known about the underlying mechanisms, and there is significantly less high-quality evidence compared to pharmacological intervention studies. Due to the immense potential of prevention in heart research, the cardiologic societies are calling for research work to be intensified, from basic understanding to clinical application. The topicality and high relevance of this research area are also evident from the fact that in March 2018, a one-week conference on this topic with contributions from top international scientists took place as part of the renowned \u201cKeystone Symposia\u201d (\u201cNew Insights into the Biology of Exercise\u201d). Consistent with the link between obesity, exercise, and cardiovascular disease, this review attempts to draw lessons from stem-cell transplantation and preventive exercise. The application of state-of-the-art techniques for transcriptome analysis has opened new avenues for tailoring targeted interventions to very individual risk factors. The underlying process that leads to the development of MI is atherosclerosis. Risk factors that favor the development of atherosclerosis include smoking, hyperlipidemia, arterial hypertension, diabetes mellitus, obesity, and a lack of exercise . The endResearch into the genetic-physiological basis of myogenesis and adipogenesis is crucial to assessing the influence of risk factors that favor the development of atherosclerosis. Commonly, inbred mouse lines are used for most studies. However, human populations are genetically diverse, so inbred lines should be viewed critically when considering the genetic contribution to the disease. The use of non-inbred mice should be promoted.low monocytes play a central role in the proliferative phase by stimulating fibroblasts, angiogenesis, and collagen formation [high and Ly-6Clow monocytes are chemotactically directed into the myocardium via the cytokine gradients of CCR2 (CC chemokine receptor type 2) or CX3CR1 (CX3C chemokine receptor 1). The Ly-6Chigh monocytes are phagocytic and proteolytically active cells that dominate the inflammatory phase.Myocardial healing in the first few weeks after acute MI is a highly dynamic and complex process that has been studied in great detail, especially in the mouse model. The biological processes occur in three characteristic phases in the hypoxic damaged murine myocardium. In the first 72 h, there is an intense inflammation dominated by infiltrating leukocytes, which clear away dead cells and the intercellular matrix. In the subsequent proliferative phase (up to approximately seven days after acute ischemia), the infarcted myocardium is replaced by granulation tissue. Fibroblasts differentiate into myofibroblasts and secrete an extracellular matrix traversed by a sprouting vascular network. In the maturation phase, fibroblasts undergo apoptosis, the angiogenetically fused vascular network recedes, and a collagen-rich scar develops [ormation . NahrendThe sprouting of blood vessels into the infarct area\u2014angiogenesis\u2014is an integral part of the healing myocardium, which is coordinated mainly by the cytokine environment of the inflammatory cells. Angiogenesis describes the de novo sprouting of capillary networks from endothelial cells in existing post-capillary venules . Tissue A skin lesion heals in a strictly organized sequence of the immune response, angiogenesis, and, ultimately, scarring. Wound healing following tissue damage is a highly conserved repair process similar to most humans and vertebrates\u2019 organs ,12. The Impaired wound healing is an important health problem. Signs of aging and diseases such as stress and diabetes, as well as obesity, can lead to a chronic inflammatory state at a low level, which may disrupt wound healing. Obesity has a significant adverse impact on the wound-healing process. The latest findings indicate that aberrant inflammation of the wound site may be the cause of delayed healing . InterveInterestingly, a comparative study showed that high-fat diet (HFD) mice did not develop any chronic wounds in contrast to genetically diabetic (ob/ob) mice. However, healing was delayed as a result . While oWound healing consists of integrated and overlapping phases of hemostasis/coagulation, inflammation, proliferation, and dissolution/remodeling. These phases must occur at defined times and last for a certain period; otherwise, pathological wound healing occurs . ChronicThe human predisposition to obesity results from the interaction between biology and culture over the course of human evolution. Hominid ancestors and modern humans were more often confronted with food shortages on the one hand and had to be very physically active on the other . Therefo2 [2) people with diabetes and non-ischemic heart disease [2) with signs of impaired contractile function have abnormally high levels of triglycerides in the heart [Paradoxically, obesity has been protective shortly after MI when associated with less comorbidity . Neverth2 . In the 2 , a conne2 . Against2 . Thakker2 . After 22 . Phenoty2 ,43. The 2 . The den2 . Lipid d disease . It is u disease ,47,48. I disease . Thus, T disease . Zhou an disease . To diff disease , peroxis disease ,51,52,53he heart . In addihe heart . Elevatehe heart ,56.The data from the Federal Statistical Office show that chronic ischemic heart disease and acute MI caused more than 35% of all deaths in Germany in 2017, in most cases due to advanced coronary artery disease. In addition to hemodynamic, metabolic, and biochemical changes, structural damage to the heart tissue also affects the electrophysiology of the heart. Such damage is most common after acute MI and results in increased arrhythmogenicity in the heart. Arrhythmias with harmful prognostic properties lead to increased therapy-relevant mortality , wherebyFurther changes are observed in the connexons. The dephosphorylation breaks the coupling of the cells of connexin 43, and the physiological conduction of excitation at the gap junctions is disturbed . While tHowever, while mortality from acute MI has decreased significantly since 1980, chronic ischemic heart disease as a secondary disease has been at the top of the list of the most common causes of death since 1992. In the early stages of the disease, pharmacological interventions can prevent progression and, if adherence to therapy is optimal, even reduce coronary stenoses . Early r+ mesenchymal stem cells with surrounding cardiomyocytes were described, which trigger the intrinsic program of the cardiogenic lineage specification of the mesenchymal stem cells [+ mesenchymal stem cells have no arrhythmogenic properties and are even a suitable cell type to prevent arrhythmias after MI [Due to the lack of causal treatment options, the last 30 years of cardiovascular research in cell transplantation have been shaped by the euphoric hope that lost heart tissue could be replaced with stem cells. Initially, the focus was on the intramyocardial transplantation of adult stem cells, which can promote specific cardiac functions and follow cardiovascular differentiation pathways but are safe to use. The therapeutic effects on the intercellular communication of CD271em cells . These wafter MI .V\u03b23 after MI [In the last 20 years, different cell populations have been investigated concerning their therapeutic effects on ischemically damaged myocardium. Almost all cell preparations had a measurable positive effect on improving pump function. To obtain an overview of the enormously extensive research field and to summarize and systematically evaluate the countless stem-cell experiments in the mouse model for the therapy of acute MI, meta-analyses were started in this area Figure . Our stuafter MI . It was after MI . In connafter MI ,70. The after MI .+ bone marrow stem cells, which is mainly based on the regulation of angiogenesis [+ stem-cell transplantation/coronary artery bypass surgery) with patients after MI and coronary heart disease, we observed a preoperative signature of circulating bone marrow stem cells and endothelial progenitor cell parameters in the peripheral blood that correlated significantly with the postoperative response of myocardial regeneration [In addition, we addressed the regenerative effectiveness of hematopoietic CD133ogenesis ,73,74,75neration . Now, foneration on the subject of cardiac stem-cell therapies , the sumCoronary artery disease is largely caused by modifiable behaviors such as physical inactivity, an unhealthy diet, and smoking . The inc2) and a waist circumference of \u226494 cm for men and \u226480 cm for women. To improve the risk profile of cardiovascular disease and reduce the threat of type 2 diabetes, overweight and obese people are advised to aim for weight reduction. Although a number of short-term diets are effective for weight loss, a healthy diet should be maintained over a longer period of time in view of the risk of cardiovascular disease. If lifestyle changes do not result in sustained weight loss, bariatric surgery and SGLT2 inhibitors and GLP-1RA to reduce future cardiovascular disease may be considered in high-risk obese patients (BMI > 40). The use of SGLT2 inhibitors and GLP-1RA is recommended for patients with coronary heart disease because of their proven prognostic benefits [The recommendations of the current ESC prevention guideline form a new basis for joint decision-making by doctor and patient in the selection of preventive measures based on individual patient characteristics. The therapy goals can be individually adapted in a step-by-step procedure. While BMI, waist circumference, and waist-to-hip ratio do not improve the prediction of cardiovascular disease risk, a comprehensive threat assessment should be considered in overweight/obese individuals. The classic BMI categories are increasingly being disaggregated to focus on waist circumference, as this is a marker of central obesity and is strongly associated with the development of CHD and diabetes. The current prevention goal is weight reduction to a normal weight (BMI < 25 kg/mbenefits .In addition, aspects of the current ESC guideline also include the use of a new risk algorithm based on cardiovascular morbidity and an adjusted risk calculation for patients with established atherosclerotic cardiovascular disease, obesity, and hypertension. For example, regular screening for sleep disorders is indicated. To reduce body weight and prevent or slow weight gain, lifestyle modifications, including smoking cessation, a low-fat, high-fiber diet, aerobic and resistance training, and reducing energy intake, are recommended for people with diabetes. In newly diagnosed diabetes mellitus, a hypocaloric diet with aggressive weight reduction can lead to remission of the disease. To this end, a low-calorie diet should be considered early after diagnosis, followed by a period of food reintroduction and a period of weight maintenance .The trend towards overweight and obesity is currently pointing upward. Approximately 8% of all deaths worldwide are attributed to obesity, with a meta-analysis from 2017 showing that even moderate weight loss reduces all-cause mortality by 18% . EffectiIn this way, a basis can be established to identify targeting mechanisms for biomarkers that represent the effect of personalized lifestyle interventions tailored to individual risk factors.Interventions that can accelerate the chances of healing for those with slow or non-healing wounds are critical. Studies show that exercise training tends to reduce inflammation in obesity, regardless of whether human or animal models are used ,90. GiveExercise is a relative intervention strategy that can be used clinically to prevent or treat disorders in the healing process. Little is known about the mechanisms by which movement accelerates healing. In addition, clinical studies on obese people are needed to determine whether the results can be transferred from obese animal models. We use the non-inbred mouse line DU6, established over 146 generations by phenotype selection for a high body mass 42 days after birth ,94, as wIn contrast, there are twice as many endothelial cells in the Fzt:DU strain as in C57BL/6 mice . Among tThe protective effect of endurance training on the cardiovascular system has long been known, but the underlying molecular mechanisms are only partially understood . In thisPotential starting points for deciphering these mechanisms arise from new findings in the area of wound healing from injuries to the skin. Due to the preserved healing processes, the basic concepts should be transferable to myocardial healing . The pheInterestingly, there is also increasing evidence that the disturbed inflammation profile and, thus, the skin healing capacity can be improved through endurance training ,92. ThatAt present, only initial data on the mechanisms underlying the cardioprotective effects of endurance training exists. A targeted search for possible biomarkers to measure the effectiveness of the lifestyle intervention sport\u2014as required by the ESC\u2014could clarify to what extent obesity plays a significant role during the healing process after MI due to the change in the cellular inflammation profile. If the individualized pathomechanisms identified in the mouse correlate with specific data from heart attack patients, the healing process after an MI could be significantly improved based on patient-specific diagnosis and targeted prediction, including treatment.After intensive research into regenerative concepts such as stem-cell therapy over the last 20 years, the healing potential remains relatively limited despite the enormous research effort. Preventive approaches to activate endogenous repair mechanisms and, thus, on the one hand, minimize the extent of damage caused by tissue ischemia and, on the other hand, improve wound healing are therefore of great interest. In the search for alternative approaches to minimize the extent of functional damage, the improvement of endogenous healing mechanisms is more important than ever. The research groups are increasingly concentrating on the connection between the chronic inflammatory state of the heart and its associated negative impact on the wound healing process. Although the cardiological societies have long agreed on the cardioprotective effect of a healthy lifestyle, there still needs to be more understanding of a real translational lifestyle intervention concept, from the pathophysiological mechanisms in animal models to the clinical manifestation of the respective disease. The aim should be to identify mechanisms for biomarkers that represent the effect of personalized lifestyle interventions tailored to individual risk factors."} +{"text": "The efficient functioning of the healthcare system is greatly dependent on nursing staff. To improve health behaviors among nurses, systemic solutions such as workplace wellness programs, incentives for healthy behaviors, and education on the benefits of a healthy lifestyle are needed.Health behaviors play a pivotal role in improving and strengthening health. Nurses, who constitute the vast majority of employees in the health sector, play a crucial role not only in treating disease but also in promoting and maintaining optimal health for themselves and society. The purpose of the study was to assess the level of health and sedentary behavior and the factors influencing them among nurses. A survey, cross-sectional study was conducted among 587 nurses. Standardized questionnaires evaluating health and sedentary behavior were used. The study utilized both single-factor and multifactor analyses, employing the linear regression method and Spearman correlation coefficient. The results showed that the health behaviors of the survey nurses were at an average level. Sedentary time (in hours) was an average of 5.62 h (SD = 1.77) and correlates significantly ( In recent years, there has been growing public awareness of the importance of health behaviors in sustaining and improving overall health ,2,3,4,5.Unfortunately, numerous studies have indicated that employees in the healthcare sector, including nurses, often fail to practice behaviors that promote and sustain good health, despite their knowledge and high awareness of health . It is dThis is not only a workplace health problem but also a potential financial and patient safety issue. Creating policies and jobs that take into account holistic support for this professional group is a guarantee of stress reduction, professional burnout, better health condition, and thus patient safety and high-quality healthcare ,23,24.The efficient functioning of the healthcare system is greatly dependent on nursing staff . Staff sThe purpose of the study was to assess the level of health and sedentary behavior and the factors influencing them among nurses.This is a cross-sectional descriptive study, carried out from April to June 2021 in the Subcarpathian region in Poland. The study involved 587 nurses working in the following medical entities: primary health care, hospitals, outpatient specialist care, hospices, independent health care institutions, health care centers and social welfare homes, private sector, nursing homes, and resorts. Medical entities have been randomly selected through a randomized algorithm program. The sample size was determined using EPI INFO software. A multistage random cluster sampling method was used.Convenient sampling was used, and the inclusion criterion for the participants was professionally active nurses with a license to practice, at least 2 years of work experience, and consent to participate in the study. The questionnaire was distributed to participants in sealed envelopes to ensure confidentiality. In total, 1150 questionnaires were distributed, 614 were collected. All questionnaires were checked, and due to the incompleteness of the answers, 27 questionnaires were excluded from the study. Data from 587 questionnaires were entered into an Excel spreadsheet, coded, and subjected to statistical analysis. To explain the purpose and conditions of the study, a meeting was organized in the medical entities before the survey. Participation in the study was voluntary, with the possibility of withdrawing from the study at any stage and without consequences. The nurses were assured of the anonymity of the study. The nurses included in the study were a representative group of all nurses working in the southeastern region of Poland .The survey technique used a standardized questionnaire, including the Health Behaviors Inventory (HBI), a modified Polish version of the International Physical Activity Questionnaire (IPAQ), and questions regarding the sociodemographic data of the respondents.The HBI questionnaire is used to monitor pro-health behaviors; it contains 24 statements describing health-related behaviors. Analysis of the frequency of individual behaviors indicates the overall result of individual health behaviors.-Proper Eating Habits (PEH), which primarily take into account the type of food eaten ;-Preventive Behaviours (PB), related to compliance with health recommendations and how to obtain information in the field of health and disease;-Health Practices (HP), related to daily habits related to sleep, physical activity, and recreation;-Positive Mental Attitude (PMA), related to psychological factors such as avoiding stress, strong emotions, or other depressing situations.The questionnaire is divided into four subscales:Respondents specifying the frequency of specific health behaviors answer questions on a five-point scale, where 1 means almost never, 2 means rarely, 3 means occasionally, 4 means often, and 5 means almost always. The questionnaire evaluates the frequency of behaviors over the last year, and the average examination time does not exceed 5 min.To obtain a general indicator of the intensity of health behaviors, all values marked by the respondent are summarized. The number of points varies from 24\u2013120. Higher scores correspond to a higher level of declared health behaviors. The results are interpreted according to the sten scale: according to the norms (separate for women and men) given in the key to this questionnaire, sten scores 1\u20134 mean low, sten scores 5\u20136 average, and sten scores 7\u201310 high regarding increased health behaviors. The results of each of the four subscales of the HBI questionnaire are the averages of the responses given to the questions included in them. The results obtained were interpreted as low, high, or medium according to the following criteria: an average of 1 means \u201calmost never\u201d; an average of 2 means \u201crarely\u201d; an average of 3 means \u201cfrom time to time\u201d; an average of 4 means \u201coften\u201d; and an average of 5 means \u201calmost always\u201d .Respondents retrospectively reported their sedentary behaviours (SB). To obtain the most accurate results, before the survey, nurses were asked to pay attention to and/or record how much time they spend sitting during the day before respondents retrospectively reported their sedentary behavior in the survey. A single item questionnaire (the short version of the IPAQ questionnaire) was used. The question asked: \u201cHow long have you been sitting in the last 7 days? When answering, nurses indicated how much time (in hours per day) they spent sitting .The analysis of quantitative, which represent numerical, data was performed by calculating the mean, standard deviation, median, and quartiles. The analysis of qualitative variables was performed by tabulating the frequency and percentage of each value. Single- and multi-factor analysis of the influence of many variables on the quantitative variable was performed using the linear regression method. The results are presented as values of the parameters of the regression model with a 95% confidence interval.p-values less than 0.05 were interpreted as correlations that are unlikely to have occurred by chance.The correlations between the quantitative variables were analysed using the Spearman correlation coefficient. The analysis adopted a significance level of 0.05. Therefore, all The analysis was performed in the R program, version 4.1.3 .The study involved 587 nurses, mostly women (80.75% vs. 19.25%), aged 38\u201352. Details are presented in The results showed that almost half (41.74%) of the participants declared an average level of health behaviors, 33.39% were high and 24.87% were low .The average results of the subscales \u201cProper eating habits\u201d, \u201cPreventive behaviors\u201d and \u201cPositive mental attitude\u201d were within the range of 4 in a scale of 1\u20135, where 4 indicates a high frequency of behaviors in these areas. The average result of the \u201cHealth practices\u201d subscale was 3.39 points (rounded to 3). Thus, the average frequency of undertaking behaviors in this area is \u201cfrom time to time\u201d .p > 0.05), showed that none of the variables analyzed in the study are a significant predictor of the score on this scale (> 0.05), .p < 0.05) predictor of health behavior among nurses was the level of education, specifically bachelor\u2019s title . The multivariate linear regression model within the scope of the PEH subscale showed that important (p < 0.05) independent predictors of the result of health behavior among nurses were the level of education, specifically the bachelor\u2019s title , and the workplace\u2014that is, work in IHI , showed that an important (p > 0.05), in the scope of the PB subscale showed that none of the variables analyzed in the study features is an important predictor of the result on this scale (p > 0.05). The multivariate linear regression model in the scope of the PMA subscale showed that important (p < 0.05) independent predictors on this scale are age and nurses\u2019 work experience , in the scope of the PMA subscale showed that none of the analyzed features are an important predictor of the result on this scale (p > 0.05), details available in The single and multivariate linear regression model in the HP scope showed that none of the variables analyzed is an important predictor of the result on this scale (The results showed that sedentary time (in terms of hours) had an average of 5.62 h (SD = 1.77) and ranged from 1.71 to 11.29 h/day .p < 0.05) predictor of sitting time among the nurses surveyed , with a regression parameter of \u22120.38. This indicates that each additional job reduces sitting time by an average of 0.38 h per day. The multivariate linear regression model showed that the gender of men is still a significant (p \u02c2 0.05) independent predictor of sitting time, with a regression parameter of \u22120.489. This means that it reduces sitting time by an average of 0.489 h per day with respect to the female sex showed that the gender of the male is a significant and negatively (r < 0) with health behaviors, specifically with the positive mental attitude subscale. This means that the longer the sitting time, the lower the intensity of this type of health behavior per day being sedentary, with a range from 1.71 to 11.29 h. These results are consistent with those of Benzo et al. who reported that nurses spent 4.56 h of each 12-h shift sitting and 5.64 h of each 12-h shift standing . The corThere are several potential limitations of this study that should be considered when interpreting the results. Firstly, the study had a limited geographic scope and there may be differences between departments of healthcare institutions. Secondly, the study was carried out during the COVID-19 pandemic, making it difficult to reach a larger group of respondents. It was also a very difficult time for nurses because they worked under difficult conditions. Thirdly, despite the assurance of anonymity, it was a self-assessment of specific health and sedentary behaviors, which may be partly subjective. Fourthly, the cross-sectional design of the study prevents us from establishing causality and temporality problems. Finally, sedentary behaviors were assessed based on the retrospective declaration of the nurses. Studies using more advanced and objective measuring devices, e.g., accelerometers, are needed. To increase the generality of the study, it should be extended and include more medical facilities in other regions.The nurses in our study presented a moderate level of health behaviors, with the highest results in terms of preventive behaviors on the HBI scale and the lowest scores in terms of health practices. Age and work experience were found to affect nurses\u2019 positive mental attitude, while level of education influenced proper eating habits. Additionally, the study found that the average sedentary behavior was 5.62 per day. To improve health behaviors among nurses, systemic solutions such as workplace wellness programs, incentives for healthy behaviors, and education on the benefits of a healthy lifestyle are needed.Nursing leaders and employers play a pivotal role and can become advocates for system changes and be aware of the impact of social support and the hospital or unit culture on the ability of a nurse to practice health-promoting behaviors. They should create open work environments where nurses feel comfortable talking about workplace stressors and barriers to healthy behaviors, and thus support and promote nurses\u2019 efforts to eat a healthy diet or exercise. When making staffing decisions, they should ensure that there is adequate support to provide meal and relaxation breaks for nurses. It should be noted whether canteens, coffee shops, and vending machines in medical facilities offer healthy food.Because nurses themselves are best able to identify the needed programs and barriers in the workplace to healthy living, it is a good initiative to assess needs or hold focus groups to determine how best to encourage and support a healthy lifestyle. Holistic activities that promote health permanently, not only periodically, focusing on physical activity, healthy eating, and stress reduction, must be included in the nursing workplace. Both mindfulness-based stress reduction interventions and weekly yoga classes can be helpful in improving self-care and reducing nurse burnout. They are essential and should be activities that staff members can perform during the workday or immediately before or after their shifts.A very positive example of initiatives taken in this area is the Healthy Nurses\u2019 Nation Health campaign created by the American Nurses Association, which created an information website especially for nurses and thus supports and promotes pro-health behaviors among nurses .Incorporating healthy behaviors into the nursing workplace is an investment that can not only provide immediate dividends for the nurses and staff, but long-term benefits for the organization and patients as well. In an age when it is imperative that all possible avenues for improving quality of care and decreasing healthcare costs be explored, interventions aimed at promoting the health, well-being, and performance of Polish nurses should be implemented to support nurses in making lifestyle changes and improving their health. Given the current shortage of nurses in Poland, this is of paramount importance. We hope that the findings of this study prompt further investigation in this area."} +{"text": "Temperature-sensitive carboxylated cellulose nanocrystals/N-isopropyl acrylamide aerogels (CCNC-NIPAMs) were developed as novel pesticide-controlled release formulas. Ammonium persulfate (APS) one-step oxidation was used to prepare bagasse-based CCNCs, and then the monomer N-isopropyl acrylamide (NIPAM) was successfully introduced and constructed into the temperature-sensitive CCNC-NIPAMs through polymerization. The results of the zeta potential measurement and Fourier infrared transform spectrum (FTIR) show that the average particle size of the CCNCs was 120.9 nm, the average surface potential of the CCNCs was \u221234.8 mV, and the crystallinity was 62.8%. The primary hydroxyl group on the surface of the CCNCs was replaced by the carboxyl group during oxidation. The morphology and structure of CCNC-NIPAMs were characterized via electron microscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), compression performance, porosity analysis, and thermogravimetric (TG) analysis. The results demonstrate that CCNC-NIPAM has a high porosity and low density, as well as good thermal stability, which is conducive to loading and releasing pesticides. In the swelling, drug loading, and controlled release process, the CCNC-NIPAM exhibited significant temperature sensitivity. Under the same NIPAM reaction amount, the equilibrium swelling rate of the CCNC-NIPAM first increased and then decreased with increasing temperature, and the cumulative drug release ratio of the CCNC-NIPAM at 39 \u00b0C was significantly higher than that at 25 \u00b0C. The loading efficiency of the CCNC-NIPAM on the model drug thiamethoxam (TXM) was up to 23 wt%, and the first-order model and Korsmyer\u2013Peppas model could be well-fitted in the drug release curves. The study provides a new method for the effective utilization of biomass and pesticides. As the most abundant natural material, cellulose has the characteristics of being renewable, biodegradable, non-toxic, abundant reserves, biocompatible, low cost, and so on. Bagasse is an important by-product of the sugar industry, and in recent years, the global annual production of bagasse has reached 260 million tons . How to The preparation process of CCNCs focuses on reducing the size of cellulose, removing the amorphous region in cellulose as far as possible ,7,8, andTo deal with the threat posed by the overuse of pesticides to the environment and human beings, researchers have proposed pesticide controlled release preparations as a solution. Most pesticide active ingredients are hydrophobic organic compounds that require the addition of carriers, solvents, emulsifiers, dispersants, or other auxiliary ingredients to make them easy to use in the field. Thiamethoxam (TXM) belongs to the second generation of nicotinic insecticides, which includes a wide range of insecticidal species that have tactile and gastric toxic effects on pests, high stability, and are easily decomposed. It was used as a model drug in this study.Aerogel is a kind of three-dimensional nanostructured porous material composed of nanoparticles or polymer molecular chains that has the structural characteristics of low density, high porosity, high pore volume, and high specific surface area. Environmentally responsive aerogels can stimulate and respond to changes in the environment such as light, pH, and temperature and are widely used as carriers for drug controlled release preparations . ResearcTo prepare pesticide controlled release preparations with ideal mechanical and drug release properties, the physicochemical properties of the complexes can be improved by adding nano-additives to the polymer skeleton . Due to In this study, bagasse was used as a raw material to prepare CCNCs through APS one-step oxidation, and then NIPAM was introduced. Under the action of a crosslinking agent and initiator, a temperature-sensitive nanocellulose aerogel (CCNC-NIPAM) was formed. The morphology and structure of the CCNC-NIPAM were analyzed with Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), thermogravimetric (TG) analyses, and electron microscopy. A single-factor experiment was designed to adjust the structure and properties of the gel. TXM was used as the model drug to study the performance of the aerogel in the subsequent drug loading and controlled release experiment. In addition, the existing zero-order model, first-order model, Higuchi model, and Peppas model were used to fit the release curves to explore the drug release mechanism.Bagasse was obtained from Guangxi, China . Ammonium persulfate was purchased from Guangdong Guanghua Technology Co., Ltd. . N-isopropyl acrylamide and thiamethoxam were purchased from Macklin Co., Ltd. . N, N\u2032-methylene diacrylamide and phosphate buffered saline (PBS) were purchased from the Sinopharm Group . All chemicals used in this study were used after receipt and were not further purified.Bagasse was put into a planetary ball mill tank for ball milling treatment for 20 min; 1.5 g of powder was weighed and added into 150 mL of APS solution, and then stirred at 500 r/min at 60 \u00b0C. After the reaction for several hours, the product was centrifuged several times at 10,000 r/min until the supernatant was a stable light blue CCNC suspension. The precipitation and supernatant after centrifugation were retained, and an ultrasound was performed after appropriate dilution. The bulk precipitates were dispersed and put into dialysis bags for 3 days. The supernatant was kept and stored in a refrigerator at 4 \u00b0C for later use. Some of the suspension was lyophilized to obtain CCNCs in powder form.2. Three hours after the reaction, 200 mL of pre-cooled deionized water was added to the flask to terminate the reaction, and the solvent was changed periodically to remove the unreacted monomer. After the solution was clarified, the product was immersed in anhydrous ethanol for 8 h, and finally, the product was freeze-dried in a vacuum freeze-drying instrument for 48 h. Three kinds of temperature-sensitive aerogels were designated as CCNC-1NIPAM, CCNC-2NIPAM, and CCNC-3NIPAM, with a mass ratio of NIPAM to CCNC of 1:1, 1.5:1, and 2:1, respectively.A certain amount of CCNCs and NIPAM was weighed and dissolved in deionized water. N, N\u2032-methylene diacrylamide (MBA) was used as the crosslinking agent, and potassium persulfate was used as the initiator. The mixture was evenly mixed under the protection of NScanning electron microscopy (SEM) images were acquired using Hitachi SU8220 (Japan) at an accelerating voltage of 10 kV.The sample powders were analyzed on a Fourier transform infrared spectroscope , followed by mixing with KBr (1:100), and then subsequently pressed into a disk. Samples were dried overnight in a vacuum oven at 60 \u00b0C before measurements were taken.I002 is the strong diffraction peak near 22.4\u00b0 and Iam is the strong diffraction peak in the amorphous region (the highest diffraction intensity between I002 and I110 is used here).Wide-angle X-ray diffraction was used to evaluate the crystallinity of the samples. The measured voltage was 40 kV, and the current was 100 mA. The Segal empirical formula [Equation (1)] was used to calculate the crystallinity of cellulose (Cr. I), where CCNCs and the CCNC-NIPAM were characterized by K-Alpha X-ray photoelectron spectroscopy in the range of 0~700 eV, and the data were processed by the software provided with the device.The compression performance of the CCNC-NIPAM was determined using a universal testing machine at a compression rate of 10 mm/min, and the rod-like sample deformation rate was set at 80% without cyclic testing.2 protection. The heating rate was 10 \u00b0C/min, and the temperature range was 30~800 \u00b0C.The thermal stability and decomposition temperature of the CCNCs and CCNC-NIPAM were characterized via a thermogravimetric analysis. Then, 2~10 mg of CCNCs and CCNC-NIPAM were weighed and tested by a thermogravimetric analyzer under NThe size of the particle and the charge it carries on the surface of the sample in suspension (0.01 wt%) were determined at 25 \u00b0C using a nano-zetasizer device . The average zeta potential was recorded for 5 measurements, each consisting of 12 repeated runs.The prepared CCNC was characterized by ADVANCE NEO 400WB NMR spectrometer .1 is the total mass of the freeze-dried bottle and sample CCNCs after drying, in g; m2 is the mass of the pre-weighed freeze-dried bottle, in g; V is the total volume of CCNC suspension obtained, in mL; and m is the mass of bagasse cellulose treated by APS oxidation, in g.The total volume of CCNC suspension was accurately measured, and 30 mL of suspension was taken with a pipette and placed in a freeze-dried bottle that had been dried and weighed. The suspension was frozen at \u221240 \u00b0C and then placed in a vacuum freeze-drying instrument. After the samples were completely dried to constant weight, they were collected and weighed, and the concentration of suspension was calculated according to the weight of the CCNCs. The yield of CCNCs is calculated by Equation (2).2 \u2212 V1 is the volume of NaOH solution consumed by the carboxyl group on the CCNCs, in mL.In this study, conductance titration was used to determine the oxidation degree of CCNCs. Firstly, 0.2500 g of CCNC powder was added into a beaker, and 50 mL of 0.01 mol/L HCl solution was added. After mixing evenly, the CCNC suspension was titrated with 0.01 mol/L sodium hydroxide (NaOH) solution, and the volume and corresponding potential of the NaOH solution were recorded. The calculation formula for carboxyl group content in CCNCs is shown in Equation (3).1 is the total mass of aerogel and ethanol, in g; m0 is the dry weight of aerogel before soaking, in g; \u03c1 is the density of ethanol, in g/cm3; and V is the volume of the aerogel sample, in cm3.The porosity of the CCNC-NIPAM was determined using the liquid displacement method. Ethanol is used as the replacement liquid because ethanol will not cause an expansion or contraction when permeating into the thermosensitive nanocellulose aerogel. A certain volume of freeze-dried CCNC-NIPAM was cut, its mass was accurately weighed, and the aerogel was immersed in ethanol for 24 h, then suspended for 20 s to remove excess ethanol from the surface of the aerogel and weighed. The calculation formula is as Equation (4).1 is the mass of aerogel after swelling, in g, and W0 is the initial mass of the aerogel, g.The swelling ratio (SR) of the CCNC-NIPAM was determined. A certain amount of aerogel was weighed and immersed in 30 mL of PBS solution at different temperatures and stirred. The expanded gel was removed from the solution at regular intervals and then weighed after standing in the air for 20 s. All experiments were repeated three times. The SR is calculated by Equation (5).t is the gel mass measured at the time during the swelling elimination process, and me is the gel swelling equilibrium mass).CCNC-1NIPAM, CCNC-2NIPAM, and CCNC-3NIPAM, which reached equilibrium in deionized water at 25 \u00b0C, were placed in constant-temperature water at 60 \u00b0C. The gel shrunk and produced swelling changes due to heat. After removing the gel at regular intervals, the excess water was wiped off the surface of the gel with filter paper, and the gel was weighed until it was constant. The degree of detumescence is calculated by Equation (6):0 is the TXM mass loaded on aerogel, in g; m1 is the aerogel mass, in g; and m2 is the TXM mass input in the initial drug loading solution, in g.The spare aerogel was stirred at 500 r/min in solutions with different initial concentrations of TXM, and then the aerogel loaded with the drug was removed and lyophilized. To calculate the loading efficiency (LE) and encapsulation efficiency (EE) of the aerogel on the TXM, a series of TXM standard solutions with a concentration gradient were configured. The absorbance of the solution at 251 nm was scanned and measured using a UV\u2013visible spectrophotometer to generate a calibration curve. The drug-loaded aerogel was ground into powder and weighed accurately. The samples were dispersed in phosphate buffers at different temperatures . After stirring at 700 r/min for 24 h, the TXM content in the supernatant was determined using the peak at 251 nm as the specific absorption peak. The LE and EE are calculated by Equations (7) and (8). All tests were repeated 3 times, and the averages were recorded.e is the volume of solution extracted within a predetermined time interval, in mL; V0 is the initial volume of the solution, in mL; \u2211Ci is the sum of drug concentrations released from sample to buffer at sampling interval 1 to n \u2212 1, in g/mL; Cn is the drug concentration released at the NTH sampling interval, in g/mL; and mDrug is the weight of TXM, in g.Drug release is a complex phenomenon that occurs through a diffusion mechanism. In this study, the CCNC-3NIPAM with the highest LE was selected and put into 200 mL of the PBS solution at 25 \u00b0C and 39 \u00b0C, respectively, for the drug release study to explore the relationship between the temperature and drug release behavior of the gel. To evenly distribute the released TXM in the PBS solution, the PBS solution was oscillated in a constant-temperature shaker at a rate of 200 r/min. Different time intervals were set, and 1.0 mL of supernatant was extracted for analysis. To keep the total solution volume constant, an equal amount of fresh solution (1.0 mL) was added. The absorbance of TXM in the solution at 251 nm was measured using a UV\u2013visible spectrophotometer and compared with the standard curve to calculate the cumulative release of TXM. The cumulative drug release is calculated by Equation (9):0, k1, k2, and k3 are the release rate constants of the zero-order, first-order, Higuchi, and Korsmeyer\u2013Peppas models, respectively. F is the drug release quantity at time t, and n is the diffusion or release index representing the drug release mechanism. The most suitable model for the experimental data is selected according to the comparative correlation coefficient.To further clarify the kinetics of CCNC-NIPAM, the xero-order model, first-order model, Higuchi model, and Korsmeyer\u2013Peppas model Equations (10)\u2013(13) were used to fit the drug release curve. The mechanism of TXM release from aerogel was investigated under all simulated temperature conditions.The yield and oxidation degree (DO) of the CCNCs from the bagasse treated with the 1.00 mol/L, 1.25 mol/L, 1.50 mol/L, 1.75 mol/L, and 2.00 mol/L APS solutions for 16 h, 24 h, 32 h, 40 h, and 48 h were tested (no CCNCs were produced after 1 mol/L APS solution treatment for 16 h).The degree of oxidation was used to evaluate the level of the oxidation reaction and the number of groups replaced on the CCNC surface. In + and K+ ions in the PBS solution will destroy the thickness of the double electric layer of the negatively charged solute and weaken the repulsion force between ions. Therefore, it is necessary to introduce a carboxyl group on the surface of nanocellulose to reduce the content of the active hydroxyl group.The particle size test results of the CCNCs (treated with 2 mol/L APS for 24 h) are shown in 13C NMR spectra of the CCNCs. The absorbed signals of the samples are mainly at the chemical shift range of \u03b4 = 50~120 ppm, showing a typical cellulose nuclear magnetic signal absorption peak. The absorption signals at the chemical shifts \u03b4 = 68.28, 91.85, and 107.19 ppm correspond to the crystalline regions C6, C4, and C1, while the non-crystalline regions C6 and C4 are located at \u03b4 = 65.48 and 86.76 ppm, respectively. The strong absorption peaks between \u03b4 = 70 and 81 ppm are attributed to C2, C3, and C5, which are not connected to the carbon ring by the glycoside bonds. In addition, the CCNCs showed a peak at the chemical shift \u03b4 = 181.18 ppm, corresponding to the chemical shift of the carbonyl group (C=O). The results indicated that carboxylated nanocellulose was obtained after the oxidative degradation of the CCNCs with persulfuric acid, which was mainly caused by the oxidation of the hydroxyl group on C6 by H2O2 produced by persulfuric acid decomposition in the degradation process.The SEM images of the bagasse It can be concluded that the amount of NIPAM plays an important role in the structure of the aerogel, and the network structure and aperture size of the aerogel can be adjusted by changing the amount of NIPAM. The thermosensitive phase transition of CCNC-NIPAMs is caused by a change in the hydrophilic/hydrophobic balance of the cross-linked network under external conditions. XPS is an important surface analysis method that can provide element content data for a sample surface. According to the XPS spectra of the CCNCs, no signal was shown in the N1s region, which proved that there was no N element in the CCNCs. After the CCNCs were polymerized with NIPAM, the characteristic peak of nitrogen appeared at 399.1 eV in the CCNC-NIPAM spectra. The characterization results show that the N content of CCNC-NIPAM temperature-sensitive cellulose aerogels was up to 9.03% , and theThe thermal characteristics of CCNCs, CCNC-1NIPAM, CCNC-2NIPAM, and CCNC-3NIPAM were investigated and compared 2, \u2013COOH, and \u2013OH leads to an increase in the compressive stress from 15.10 kPa of CCNC-1NIPAM to 58.73 kPa of CCNC-3NIPAM.It is highly imperative to investigate the compression properties of porous aerogel with an elastic structure, as this reflects the composition and microstructure of aerogel to a certain extent. The synthesis mechanism of the CCNC-NIPAM is shown in \u22121 and C\u2013H bending and \u2013CH2 bending vibration peaks at 1375 cm\u22121, 1460 cm\u22121, 1173 cm\u22121, and 1121 cm\u22121. There is a C\u2013O\u2013C asymmetric stretching vibration at 1062 cm\u22121, at the plane stretching peak of the C=O bond [\u22121 [\u22121 and 1655 cm\u22121. The presence of these peaks indicated the successful cross-linking of the CCNC and NIPAM [\u22121, the absorption characteristic peak of \u2013NO2 at 1520 cm\u22121, and the unsaturated sulfide bond peak at 1260 cm\u22121 appeared. These characteristic peaks were attributed to TXM, indicating that TXM was successfully loaded on the aerogel.C=O bond . After tbond [\u22121 , and thend NIPAM . In FiguGenerally, the LCST of NIPAM is 32 \u00b0C. On this basis, the swelling degree (a), swelling rate (c), and equilibrium swelling degree (b) of the CCNC-NIPAM in a temperature range of 25\u201339 \u00b0C under different conditions were studied, as well as the changes in the equilibrium swelling rate of the aerogel in a PBS solution with increasing temperature (d), as shown in 1/2), if the swelling process of the material takes the diffusion of water molecules as the leading step, the SR is proportional to t1/2; otherwise, the relationship curve between SR and t1/2 is S-shaped. According to the calculated results [The swelling ratio (SR) of the CCNC-NIPAM in the solutions at different temperatures first increased and then gradually stabilized over time results c, the sw results . The pha results d. Among results .The initial concentration of the drug-loaded solution was set as 30 wt%, 50 wt%, and 70 wt% (compared with the mass of the CCNC-NIPAM). The test results of the drug-loaded solution are shown in At different temperatures, the cumulative release rate of the TXM-loaded aerogel in the PBS solution gradually increased over time. The initial burst effect of the CCNC-NIPAM in In 2 greater than 0.9444. In the Korsmeyer\u2013Peppas model, the parameter \u201cn\u201d is an important parameter for the release mechanism. When n is less than 0.45, the release mechanism of the CCNC-NIPAM is Fick diffusion. When 0.45 < n < 0.89, the release mechanism of the CCNC-NIPAM is non-Fick diffusion. When n > 0.89, drug release is mainly due to the erosion of the carrier skeleton. In this study, the fitting parameter, \u201cn\u201d, of aerogel drug release was always less than 0.45, indicating that the drug release in the CCNC-NIPAM was mainly based on the diffusion mechanism.In this study, CCNCs were successfully prepared from bagasse, and the temperature-sensitive monomer NIPAM was introduced to prepare a CCNC-NIPAM, which was applied in the field of pesticide controlled release. The ammonium persulfate one-step oxidation method has the characteristics of using low-purity materials, having a simple operation, and using high-crystallinity products. The average particle size of the prepared CCNCs is 120.9 nm, and the average surface potential of the CCNCs is \u221234.8 mV. The porosity of the CCNC-NIPAM exceeded 85%, with a highly porous structure and rich functional groups. After loading the model drug, TXM, the CCNC-NIPAM became an ideal drug release carrier and showed obvious temperature responsiveness. With the increasing mass ratio of NIPAM to CCNCs, the degree of cross-linking of CCNC-NIPAM increased, which changed the mechanical properties and porosity and also had a certain effect on the swelling behavior, and the cumulative release rate of TXM decreased during the same time. Specifically, at 25 \u00b0C, the cumulative release rate of CCNC-1NIPAM reached 76.46 wt% within 24 h, while that of CCNC-3NIPAM was only 40.55 wt%. At 39 \u00b0C, the cumulative release rate of CCNC-1NIPAM reached 86.10 wt% within 24 h, while that of CCNC-3NIPAM was only 55.06 wt%. The results show that the release kinetic fitting curves are more consistent with the first-order kinetic model and the Korsmeyer\u2013Peppas model. The purpose of this study was to explore a way to make full use of solid waste bagasse and to provide a reference for research on TXM delivery formulas." \ No newline at end of file