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10.1101/2022.05.03.489489 | A comparison of mtDNA deletion mutant proliferation mechanisms | In this paper we use simulation methods to investigate the proliferation of deletion mutations of mitochondrial DNA in neurons. We simulate three mtDNA proliferation mechanisms, namely, random drift, replicative advantage and vicious cycle. For each mechanism, we investigated the effect mutation rates have on neuron loss within a human host. We also compare heteroplasmy of each mechanism at mutation rates that yield the levels neuron loss that would be associated with dementia. Both random drift and vicious cycle predicted high levels of heteroplasmy, while replicative advantage showed a small number of dominant clones with a low background of heteroplasmy. | systems biology |
10.1101/2022.05.03.490541 | Enhanced cognitive interference during visuomotor tasks may cause eye-hand dyscoordination | In complex visuomotor tasks, such as cooking, people make many saccades to continuously search for items before and during reaching movements. These tasks require use of short-term memory and task-switching (e.g., switching search between vegetables and spices). Cognitive load may affect visuomotor performance by increasing the demands on mental processes mediated by the prefrontal cortex, but mechanisms remain unclear. It is also unclear how patients with neurological injuries, e.g., stroke survivors, manage greater cognitive loads during visuomotor tasks. Using the Trail-Making Test, we have previously shown that stroke survivors make many more saccades, which are associated limb movements that are less smooth and slower. In this test, participants search for and make reaching movements towards twenty-five numbers and letters. It has a simple variant (Trails-A), and a cognitively challenging variant (Trails-B) that requires alphanumeric switching. The switching makes the task gradually harder as the Trails-B trial progresses (greater cognitive load). Here, we show that stroke survivors and healthy controls made many more saccades and had longer fixations as the Trails-B trial progressed. In addition, reaching speed slowed down for controls in Trails-B. We propose a mechanism where enhanced cognitive load may reduce inhibition from the prefrontal cortex and disinhibit the ocular motor system into making more saccades. These additional saccades may subsequently slow down motor function by disrupting the visual feedback loops used to control limb movements. These findings augment our understanding of the mechanisms that underpin cognitive interference dynamics when visual, ocular, and limb motor systems interact in visuocognitive motor tasks.
NEW & Noteworthyo We used a neuropsychological test called the Trails-Making-test and analyze patterns of eye and reaching movements in controls and stroke survivors. We characterized how gaze and reaching movements change within a trial in the easier Trails-A and the more cognitively challenging Trails-B variant that requires alphanumeric switching. We found that as the Trails-B trial progressed participants made more saccadic eye movements and longer fixations, likely because of greater cognitive load. | neuroscience |
10.1101/2022.05.03.490527 | Hybrid offspring of C57BL/6J mice exhibit improved properties for neurobehavioral research | C57BL/6 is the most commonly used mouse strain in neurobehavioral research, serving as a background for multiple transgenic lines. However, C57BL/6 exhibit behavioral and sensorimotor disadvantages that worsen with age. We bred FVB/NJ females and C57BL/6J males to generate first-generation hybrid offspring, (FVB/NJ x C57BL/6J)F1. The hybrid mice exhibit reduced anxiety-like behavior, improved learning, and enhanced long-term spatial memory. In contrast to both progenitors, older hybrids maintain sensorimotor performance and exhibit improved long-term memory. The hybrids are larger than C57BL/6J, exhibiting enhanced running behavior on a linear track during freely-moving electrophysiological recordings. Hybrids exhibit typical rate and phase coding of space by CA1 pyramidal cells. Hybrids generated by crossing FVB/NJ females with transgenic males of a C57BL/6 background support optogenetic neuronal control in neocortex and hippocampus. The hybrid mice provide an improved model for neurobehavioral studies combining complex behavior, electrophysiology, and genetic tools readily available in C57BL/6 mice. | neuroscience |
10.1101/2022.05.03.490558 | The soil microbiome may offer solutions to ginger cultivation | The Taitung region is one of Taiwans main places for ginger agriculture. Due to issues with disease and nutrient, farmers cannot use continuous cropping techniques on ginger, meaning that the ginger industry is constantly searching for new lands. Continuous cropping increases the risk of infection by Pythium myriotylum and Ralstonia solanacearum, which cause soft rot disease and bacterial wilt, respectively. In addition, fertilizer additives cannot recover the soil when using continuous cropping on ginger, even when there is no decrease in trace elements observed in the soil. Although there may be other reasons for the reduction in production, such as soil microbes, we know little about the soil microbiome associated with ginger cultivation. Hence, in this study, we used the bacterial 16S V3-V4 hypervariable region of the 16S ribosomal RNA region to investigate microbe compositions in ginger soil to identify the difference between ginger soil with and without disease. Later, to investigate the influence of the well-known biocontrol agent-B. velezensis and fungicide Etridiazole on soil microbes and ginger productivity, we designed an experiment that collected the soil samples according to the different ginger cultivation periods to examine the microbial community dynamics in the rhizome and bulk soil. We demonstrated that B. velezensis is beneficial to ginger reproduction and suggest that it may influence the plant by adjusting its soil microbial composition. Etridiazole, on the other hand, may have some side effects on the ginger or beneficial bacteria in the soils, inhibiting ginger reproduction. | microbiology |
10.1101/2022.05.03.490561 | Antimicrobial susceptibility testing using MYCO test-system and MIC distribution of 8 drugs against clinical isolates from Shanghai of Nontuberculous Mycobacteria | Given the increased incidence and prevalence of nontuberculous mycobacteria (NTM) diseases and the natural resistance of NTM to multiple antibiotics, in vitro susceptibility testing of different NTM species against drugs from the MYCO test system and new applied drugs are required. 241 NTM clinically isolates were under analyzed, including 181 slowly growing mycobacterium (SGM) and 60 rapidly growing mycobacterium (RGM). The Sensititre SLOMYCO and RAPMYCO panels were used for the drug susceptibility testing to commonly used anti-NTM antibiotics. Furthermore, Minimum inhibitory concentration (MIC) distributions were determined against 8 potential anti-NTM drugs, including vancomycin (VA), bedaquiline (BDQ), delamanid (DLM), faropenem (FAR), meropenem (MPM), clofazimine (CFZ), avibactam (CAZ), and Cefoxitin (FOX) and epidemiological cut-off values (ECOFFs) were analyzed using ECOFFinder. The results showed that most of the SGM strains were susceptible to clarithromycin (CLA), rifampicin (RFB) from the SLOMYCO panels and BDQ, CFZ from the 8 applied drugs, while, RGM strains were susceptible to tigecycline (TGC) from the RAPMYCO panels and also BDQ, CFZ. The ECOFF values of CFZ were 0.25g/ml, 0.25g/ml, 0.5g/ml, and 1g/ml for M. kansasii, M. avium, M. intracellulare, and M. abscessus, respectively, and BDQ was 0.5g/ml for the same four prevalent NTM species. Due to the weak activity of the other 6 drugs, no ECOFF was determined. This study on the susceptibility of NTM includes 8 potential anti-NTM drugs and a large sample size of Shanghai clinical isolates. and demonstrated that BDQ and CFZ had efficient activities against different NTM species in vitro, which can be applied for the treatment of NTM diseases. | microbiology |
10.1101/2022.05.03.490560 | Subtyping evaluation of Salmonella Enteritidis using SNP and core genome MLST with nanopore reads | Whole genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing such as Oxford Nanopore Technologies (ONT) presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. However, evaluation, standardization and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of ONT for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths under seven hours of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms. | microbiology |
10.1101/2022.05.03.490559 | Large-Scale, Multi-Year Microbial Community Survey of a Freshwater Trout Aquaculture Facility | Aquaculture is an important tool for solving growing worldwide food demand, but infectious diseases of the farmed animals represent a serious roadblock to continued industry growth. Therefore, it is essential to understand the microbial communities that reside within the built environments of aquaculture facilities to identify reservoirs of bacterial pathogens and potential correlations between commensal species and specific disease agents. Here, we present the results from three years of sampling a commercial rainbow trout aquaculture facility. The sampling was focused on the early-life stage hatchery building and included sampling of the facility source water and outdoor production raceways. We observed that the microbial communities residing on the abiotic surfaces within the hatchery were distinct from those residing on the surfaces of the facility water source as well as the production raceways, despite similar communities in the water column at each location. Within the hatchery building, most of the microbial classes and families within surface biofilms were also present within the water column, suggesting that these biofilms are seeded by a unique subgroup of microbial taxa from the water. Lastly, we detected a common fish pathogen, Flavobacterium columnare, within the hatchery, including at the source water inlet. Importantly, the relative abundance of this pathogen was correlated with clinical disease. Our results characterized the microbial communities in an aquaculture facility, established that the hatchery environment contains a unique community composition, and demonstrated that a specific fish pathogen resides within abiotic surface biofilms and is seeded from the natural source water.
ImportanceThe complex microbial consortium residing in the built environment of aquaculture facilities is poorly understood. In this study, we provide a multi-year profile of the surface- and water-associated microbial communities of this biome. The results demonstrated that distinct community structures exist in the water and on surfaces. Furthermore, it was shown that a common and economically impactful bacterial pathogen, F. columnare, is continually introduced via the source water, is widespread within surface biofilms in the hatchery environment, and is likely amplified within these raceways but does not always cause disease despite being present. These results advance our understanding of pathogen localization at fish farms, show the interplay between host and environmental microbiomes, and reveal the importance of microbial community sequencing in aquaculture for identifying potential beneficial and harmful microbes. This study adds to the aquaculture microecology dataset and enhances our ability to understand this environment from a "One Health" perspective. | microbiology |
10.1101/2022.05.04.490601 | Cyclic nucleotide-induced superhelical structure activates a bacterial TIR immune effector | Cyclic nucleotide signalling is a key component of anti-viral defence in all domains of life, from bacteria to humans. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway 1, which originated in bacteria 2. These defence systems require a sensor domain such as STING or SAVED to bind the cyclic nucleotide, coupled with an effector domain that causes cell death when activated by destroying essential biomolecules 3. One example is the TIR (Toll/interleukin-1 receptor) domain, which degrades the essential cofactor NAD+ when activated in response to pathogen invasion in plants and bacteria 2,4,5 or during nerve cell programmed death 6. Here, we show that a bacterial anti-viral defence system generates a cyclic tri-adenylate (cA3) signal which binds to a TIR-SAVED effector, acting as the "glue" to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organise and complete a composite active site, allowing NAD+ degradation. Our study illuminates a striking example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation. | biochemistry |
10.1101/2022.05.04.490597 | In search of the universal method: a comparative survey of bottom-up proteomics sample preparation methods | Robust, efficient and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging, since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here we present a comprehensive comparison of 16 most widely used sample preparation methods, covering in-solution digests, device-based methods, as well as commercially available kits. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependencies and low levels of artifact formation. However, we revealed method-dependent differences in the recovery of specific protein features, which we summarized in a descriptive guide-matrix. Our work thereby provides a solid basis for the selection of MS sample preparation strategies for a given proteomics project. | biochemistry |
10.1101/2022.05.04.490694 | Huntingtin turnover: Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550 | Huntingtons disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells. | biochemistry |
10.1101/2022.05.04.490486 | Combination therapy targeting inflammasome and fibrogenesis alleviates inflammation and fibrosis in a zebrafish model of silicosis | Silicosis is a long-term lung disease caused by the inhalation of large amounts of crystalline silica dust. As there is no effective treatment available, patients are provided with supportive care, and some may be considered for lung transplantation. There is therefore an evident need for a better understanding of the diseases biology and for identifying new therapeutic targets and therapies. In this context, our group has developed a larval zebrafish model of silicosis by injecting silica crystals into the hindbrain ventricle, a cavity into which immune cells can be recruited and that mimics the alveolar environment of the human lung. The injection of silica crystals into this cavity led to the initiation of local and systemic immune responses driven through both TLR- and inflammasome-dependent signaling pathways, followed by fibrosis, as happens in human patients. The combination of the inflammasome inhibitor VX-765 and the antifibrotic agent pirfenidone was found to be the best therapy to alleviate both inflammation and fibrosis. The zebrafish model of silicosis developed here is a unique tool that will shed light onto the molecular mechanisms involved in the progression of this devastating disease and for identifying novel drugs that improve the quality of life of silicosis patients. | immunology |
10.1101/2022.05.04.490610 | Dual mode of PGRP-LE-dependent NF-κB pathway activation in bacteria infected guts | Some of the interactions between prokaryotes and eukaryotes are mediated by a molecular dialogue between microbial MAMPs and host PRRs. Bacteria-derived peptidoglycan, which possesses all the characteristics of a MAMP, is detected by membrane-bound or cytosolic PRRs belonging to various families of proteins in both animals and plants (PGRP, Nod, Lys-M...). If the identity and the epistatic relationship between the downstream components of the signaling cascades activated upon PGN/PRR interactions are well characterized, little is known about the subcellular events requires to translate these early sensing steps into downstream target gene transcription. Using a model of Drosophila enteric infection, we show that gut-associated bacteria can induce PGRP-LE intracellular aggregation. Observed in both enterocytes and entero-endocrine cells, these aggregates were found to co-localize with the early endosome marker Rab5. In vivo functional analysis further demonstrates that, whereas some PGRP-LE target genes, such as antimicrobial peptides, can be activated independently of Rab5, other such as the PGRP-SC1 amidase, need the combine action of PGRP-LE and Rab5 to be transcribed. These results demonstrate how by using different intracellular signaling routes, the same ligand/receptor complex can activate different target genes in the same cell. | immunology |
10.1101/2022.05.04.490632 | Mapping the T cell repertoire to a complex gut bacterial community | Certain bacterial strains from the microbiome induce a potent, antigen-specific T cell response1-5. However, the specificity of microbiome-induced T cells has not been explored at the strain level across the gut community. Here, we colonize germ-free mice with a complex defined community (97 or 112 bacterial strains) and profile T cell responses to each strain individually. Unexpectedly, the pattern of T cell responses suggests that many T cells in the gut repertoire recognize multiple bacterial strains from the community. We constructed T cell hybridomas from 92 T cell receptor (TCR) clonotypes; by screening every strain in the community against each hybridoma, we find that nearly all of the bacteria-specific TCRs exhibit a one-to-many TCR-to-strain relationship, including 13 abundant TCR clonotypes that are polyspecific for 18 Firmicutes in the community. By screening three pooled bacterial genomic libraries against 13 pooled hybridomas, we discover that they share a single target: a conserved substrate-binding protein (SBP) from an ABC transport system. Treg and Th17 cells specific for an epitope from this protein are abundant in community-colonized and specific-pathogen-free mice. Our work reveals that T cell recognition of Firmicutes is focused on a widely conserved cell-surface antigen, opening the door to new therapeutic strategies in which colonist-specific immune responses are rationally altered or redirected. | immunology |
10.1101/2022.05.04.490617 | Role of High Mobility Group B protein HmbA, orthologue of yeast Nhp6p, in Aspergillus nidulans | The mammalian HMGB1 protein belongs to the high-mobility-group B (HMG-B) family, which is not only architectural but also functional element of the chromatin. The fungal counterpart of HMGB1 was identified in Saccharomyces cerevisiae as Nhp6p and the pleiotropic physiological functions of this protein were thoroughly studied during the last decades. Although filamentous Ascomycete fungi also possess the orthologues of Nhp6p, their physiological functions, apart from their role in the sexual development, have not been investigated, yet. Here we study the physiological functions of the Nhp6p orthologue HmbA from Aspergillus nidulans in the primary and secondary metabolism, stress tolerance, hypha elongation and maintenance of polarized growth through the analysis of hmbA deletion mutant. We also revealed that the endochitinase ChiA acts in the cell wall remodelling and contributes to polar growth. Additionally, by conducting heterologous expression studies, we further demonstrated that HmbA and Nhp6p is interchangeable for several functions. We hypothesized that the fully complemented functions might predominantly depend on the DNA binding ability of the HmbA and Nhp6p proteins rather than on the interaction of these HMG-B proteins with other functional protein components of the chromatin. | microbiology |
10.1101/2022.05.04.490422 | Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids | Prime editing (PE) is currently the most effective and versatile technology to make targeted alterations in the genome. Several improvements to the PE machinery have recently been made, and have been tested in a range of model systems, including immortalized cell lines, stem-cells and animal models. While nick RNA (ncRNA)-dependent PE systems like PE3 and PE5 are currently considered to be the most effective, they come with undesired indels or SNVs at the edit locus. Here, we aimed to improve ncRNA-independent systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids, driven by a tissue-broad EF-1alpha promoter, that is especially suitable for human iPSC models, and linked to a GFP tag for fluorescent based sorting. These novel pAIO systems effectively corrected mutations observed in patients suffering from epileptic encephalopathy, including a truncating SCN1A R612* variant in HEK293T cells and a gain-of-function KCNQ2 R201C variant in patient-derived hiPSC, with edit efficiency up to 50%. We also show that introducing additional silent PAM-removing mutations can negatively influence edit efficiency. Finally, we observed an absence of genome-wide PE off-target effects at pegRNA homology sites. Taken together, our study shows an improved efficacy and accuracy of EF-1alpha driven ncRNA-independent pAIO PE plasmids in hiPSC. | molecular biology |
10.1101/2022.05.04.490422 | Efficient and accurate prime editing strategy to correct genetic alterations in hiPSC using single EF-1alpha driven all-in-one plasmids | Prime editing (PE) is currently the most effective and versatile technology to make targeted alterations in the genome. Several improvements to the PE machinery have recently been made, and have been tested in a range of model systems, including immortalized cell lines, stem-cells and animal models. While nick RNA (ncRNA)-dependent PE systems like PE3 and PE5 are currently considered to be the most effective, they come with undesired indels or SNVs at the edit locus. Here, we aimed to improve ncRNA-independent systems PE2 and PE4max by generating novel all-in-one (pAIO) plasmids, driven by a tissue-broad EF-1alpha promoter, that is especially suitable for human iPSC models, and linked to a GFP tag for fluorescent based sorting. These novel pAIO systems effectively corrected mutations observed in patients suffering from epileptic encephalopathy, including a truncating SCN1A R612* variant in HEK293T cells and a gain-of-function KCNQ2 R201C variant in patient-derived hiPSC, with edit efficiency up to 50%. We also show that introducing additional silent PAM-removing mutations can negatively influence edit efficiency. Finally, we observed an absence of genome-wide PE off-target effects at pegRNA homology sites. Taken together, our study shows an improved efficacy and accuracy of EF-1alpha driven ncRNA-independent pAIO PE plasmids in hiPSC. | molecular biology |
10.1101/2022.05.04.490622 | Nrf1 is an indispensable redox-determining factor for mitochondrial homeostasis by integrating multi-hierarchical regulatory networks | To defend a vast variety of challenges in the oxygenated environments, all life forms have been evolutionally established a set of antioxidant, detoxification and cytoprotective systems during natural selection and adaptive survival, in order to maintain cell redox homeostasis and organ integrity in the healthy development and growth. Such antioxidant defense systems are predominantly regulated by two key transcription factors Nrf1 and Nrf2, but the underlying mechanism(s) for their coordinated redox control remains elusive. Here, we found that loss of full-length Nrf1 led to a dramatic increase in reactive oxygen species (ROS) and oxidative damages in Nrf1-/- cells, and this increase was not eliminated by drastic elevation of Nrf2, even though the antioxidant systems were also substantially enhanced by hyperactive Nrf2. Further studies revealed that the increased ROS production in Nrf1-/- resulted from a striking impairment in the mitochondrial oxidative respiratory chain and its gene expression regulated by nuclear respiratory factors, called PalNRF1 and GABPNRF2. In addition to antioxidant capacity of cells, glycolysis was greatly augmented by aberrantly-elevated Nrf2, so to partially relieve the cellular energy demands, but aggravate its mitochondrial stress. The generation of ROS was also differentially regulated by Nrf1 and Nrf2 through miR-195 and/or mIR-497-mediated UCP2 pathway. Consequently, the epithelial-mesenchymal transformation (EMT) of Nrf1-/- cells was activated by putative ROS-stimulated signaling via MAPK, HIF1, NF-[Formula]B, PI3K and AKT, all players involved in cancer development and progression. Taken together, it is inferable that Nrf1 acts as a potent integrator of redox regulation by multi-hierarchical networks. | molecular biology |
10.1101/2022.05.04.490673 | Structural basis for activation of Arf1 at the Golgi complex | The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf GTPases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoEM structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests the movement of the GEF domain primes Arf1 for membrane insertion upon GTP binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1. | cell biology |
10.1101/2022.05.04.488533 | Thermal acclimation to warmer temperatures can protect host populations from both further heat stress and the potential invasion of pathogens | Phenotypic plasticity in response to shifts in temperature, known as thermal acclimation, is an essential component of the ability of a species to cope with environmental change. Not only does this process potentially improve an individuals thermal tolerance, it will also act simultaneously on various fitness related traits that determine whether a population increases or decreases in size. In light of global change, thermal acclimation therefore has consequences for population persistence that extend beyond simply coping with heat stress. This particularly important when we consider the additional threat of parasitism associated with global change, as the ability of a pathogen to invade a host population depends on both its capacity to proliferate within a host and spread between hosts, and thus the supply of new susceptible hosts in a population. Here, we use the host Daphnia magna and its bacterial pathogen Pasteuria ramosa to investigate how thermal acclimation may impact various aspects of host and pathogen performance at the scale of both an individual and the population. We independently test the effect of maternal thermal acclimation and direct thermal acclimation on host thermal tolerance, measured as knockdown times, as well as host fecundity and lifespan, and pathogen infection success and spore production. We find that direct thermal acclimation enhances host thermal tolerance and intrinsic rates of population growth, despite a decline observed for host fecundity and lifespan. Pathogens, on the other hand, faired consistently worse at warmer temperatures at the within-host scale, and also in their potential to invade a host population. Our results suggest that hosts could benefit more from warming than their pathogens, but highlight that considering both within- and between-host thermal performance, including thermal tolerance and fitness traits, is needed to fully appreciate how increasing thermal variability will impact host and pathogen populations. | ecology |
10.1101/2022.05.04.490575 | Impact of Salmonella genome rearrangement on gene expression | In addition to nucleotide variation, many bacteria also undergo changes at a much larger scale via rearrangement of their genome structure around long repeat sequences. These rearrangements result in genome fragments shifting position and/or orientation in the genome without necessarily affecting the underlying nucleotide sequence. To date, scalable techniques have not been applied to genome structure (GS) identification, so it remains unclear how extensive this variation is and the extent of its impact upon gene expression. However, the emergence of multiplexed, long-read sequencing overcomes the scale problem, as reads of several thousand bases are routinely produced that can span long repeat sequences to identify the flanking chromosomal DNA, allowing GS identification. Genome rearrangements were generated in Salmonella enterica serovar Typhi through long-term culture at ambient temperature. Colonies with rearrangements were identified via long-range PCR and subjected to long-read nanopore sequencing to confirm genome variation. Four rearrangements were investigated for differential gene expression using transcriptomics. All isolates with changes in genome arrangement relative to the parent strain were accompanied by changes in gene expression. Rearrangements with similar fragment movements demonstrated similar changes in gene expression. The most extreme rearrangement caused a large imbalance between the origin and terminus of replication and was associated with differential gene expression as a factor of distance moved towards or away from the origin of replication. Genome structure variation may provide a mechanism through which bacteria can quickly adapt to new environments and warrants routine assessment alongside traditional nucleotide level measures of variation. | genomics |
10.1101/2022.05.04.490580 | Three-dimensional envelope and subunit interactions of the plastid-encoded RNA polymerase from Sinapis alba | RNA polymerases (RNAPs) involved in gene transcription are found in all living organisms with degrees of complexity ranging from single polypeptide chains to multimeric enzymes. In the chloroplasts, the nuclear-encoded RNA polymerase and the plastid-encoded RNA polymerase (PEP) are both involved in the selective transcription of the plastid genome. The PEP is a prokaryotic-type multimeric RNAP found in different states depending on light stimuli and cell identity. One of its active states requires the assembly of nuclear-encoded PEP-Associated Proteins (PAPs) on the catalytic core, producing a complex of more than 900 kDa regarded as essential for chloroplast biogenesis. A purification procedure compatible with structural analysis was used to enrich the native PEP from Sinapis alba chloroplasts. Mass spectrometry (MS)-based proteomic analysis identified the core components, the PAPs and additional members, and coupled to cross-linking (XL-MS) provided initial structural information about the relative position of PEP subunits. Sequence alignments of the catalytic core subunits across various chloroplasts of the green lineage and prokaryotes combined with structural data show that the overall shape of the catalytic core and the residues essential for the catalytic activity are conserved. However, variations are observed at the surface of the core, some of them corresponding to PAP binding sites as suggested by XL-MS experiments. Using negative stain electron microscopy, the PEP 3D envelope was calculated. 3D classification shows that the protrusions which we attribute to the PAPs are firmly associated with the catalytic core. Overall, the shape of the S. alba PEP envelope is different from that of RNAPs. | biochemistry |
10.1101/2022.05.04.490442 | Functional brain region-specific neural spheroids for modeling neurological diseases and therapeutics screening | 3D spheroids have emerged as powerful drug discovery tools given their high-throughput screening (HTS) compatibility. Here, we established a method for generating functional neural spheroids with differentiated human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes at cell type compositions mimicking specific regions of the human brain. Recordings of intracellular calcium oscillations were used as functional assays, and the utility of this spheroids system was shown through disease modelling, drug testing, and formation of assembloids to model neurocircuitry. We developed disease models for Alzheimers and Parkinsons Disease through incorporating genetically engineered cells into spheroids, and Opioid Use Disorder by chronically treating spheroids with DAMGO. We reversed baseline functional deficits in each disease model with clinically approved treatments and showed that assembloid activity can be chemogenetically manipulated. Here, we lay the groundwork for brain region-specific spheroids as a robust functional assay platform for HTS studies. | bioengineering |
10.1101/2022.05.04.490579 | Force Field Dependent DNA Breathing Dynamics: A Case Study of Hoogsteen Base Pairing in A6-DNA | The Hoogsteen (HG) base pairing (bp) conformation, commonly observed in damaged and mutated DNA helix, facilitates DNA repair mechanism and DNA recognition by proteins and small molecules. The free energy difference of HG and Watson-Crick (WC) base pairs has been computed in previous computational studies. But, the mechanism of the conformational transition is not well understood. A detailed understanding of the process of WC to HG base pair transition can provide deeper understanding of DNA repair and recognition. In an earlier study, we explored the free energy land-scape for this process using extensive computer simulation with CHARMM36 force field for the nucleic acid. In this work, we study the impact of force field models in describing the WC to HG base pairing transition using the meta-eABF enhanced sampling, quasi-harmonic entropy calculation, and non-bonded energy analysis. The secondary structures of the both base pairing forms and the topology of the free energy landscapes were consistent over different force field models, although the relative free energy, entropy and the interaction energies tend to vary. The relative stability of WC and HG conformation is dictated by a delicate balance between the enthalpic stabilization and the reduced entropy of the structurally rigid HG structure. These findings provide a holistic view on the impact of force fields on the DNA base pair dynamics leading to the formation of HG conformation and will facilitate future computational investigations of DNA repair and recognition mechanism in physiologically relevant conditions. | biophysics |
10.1101/2022.05.04.489851 | The MIX domain is required for T6SS-mediated effector secretion | Bacteria use diverse classes of secreted polymorphic toxins to outcompete bacterial rivals. The MIX domain defines a widespread class of polymorphic type VI secretion system (T6SS) effectors, many of which are horizontally shared. Although several MIX-effectors have been investigated, the role of the MIX domain and the activity of many fused toxin domains remain unknown. Here, we use the Vibrio parahaemolyticus MIX-effector VPA1263 to demonstrate that MIX is necessary for T6SS-mediated secretion; however, we find that it is dispensable for interaction with the secreted T6SS tail tube component, VgrG. We also identify a C-terminal HNH nuclease toxin domain in VPA1263, and we demonstrate that it functions as a DNase during interbacterial competition. Finally, we identify variants of the genomic island containing vpa1263 in various vibrios, each carrying a different cargo of antibacterial T6SS effectors or anti-phage defense systems. We propose that these genomic islands are hotspots for bacterial defensive weapons. | microbiology |
10.1101/2022.05.04.490618 | Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces - Aspergillus interaction. | Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact between fungal and bacterial cells is a prerequisite for the metabolic reaction of the fungus that involves an amino acid starvation response and concomitant activating chromatin modifications at the affected biosynthetic gene clusters (BGCs). Here we show that not physical contact, but a guanidine containing macrolide, named Polaramycin B, triggers the response. This metabolite is produced constitutively by the bacterium and production does not need stimulation by a fungal co-culture. Polaramycin B freely diffuses in an aqueous environment and provokes the production of orsellinic acid above a certain concentration. In addition to orsellinic acid, the production of several other secondary metabolites was triggered by Polaramycin B. Our genome-wide transcriptome analysis showed that Polaramycin B treatment causes down-regulation of fungal genes necessary for membrane stability as well as genes involved in general metabolism and growth. A compensatory genetic response can be observed in the fungus that included up-regulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a specific chemical communication in which Polaramycin B, an antifungal metabolite constitutively produced by different Streptomycete species, leads to the production of fungal defence chemicals and to the up-regulation of genes necessary to compensate for the cellular damage caused by Polaramycin B. | microbiology |
10.1101/2022.05.04.490618 | Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces - Aspergillus interaction. | Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact between fungal and bacterial cells is a prerequisite for the metabolic reaction of the fungus that involves an amino acid starvation response and concomitant activating chromatin modifications at the affected biosynthetic gene clusters (BGCs). Here we show that not physical contact, but a guanidine containing macrolide, named Polaramycin B, triggers the response. This metabolite is produced constitutively by the bacterium and production does not need stimulation by a fungal co-culture. Polaramycin B freely diffuses in an aqueous environment and provokes the production of orsellinic acid above a certain concentration. In addition to orsellinic acid, the production of several other secondary metabolites was triggered by Polaramycin B. Our genome-wide transcriptome analysis showed that Polaramycin B treatment causes down-regulation of fungal genes necessary for membrane stability as well as genes involved in general metabolism and growth. A compensatory genetic response can be observed in the fungus that included up-regulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a specific chemical communication in which Polaramycin B, an antifungal metabolite constitutively produced by different Streptomycete species, leads to the production of fungal defence chemicals and to the up-regulation of genes necessary to compensate for the cellular damage caused by Polaramycin B. | microbiology |
10.1101/2022.05.04.490658 | Physics of Self-Assembly and Morpho-Topological Changes of Klebsiella Pneumoniae in Desiccating Sessile Droplets | We have investigated the flow and desiccation-driven self-assembly of Klebsiella Pneumoniae in the naturally evaporating sessile droplets. Klebsiella Pneumoniae exhibits extensive changes in its morphology and forms unique patterns as the droplet dries, revealing hitherto unexplored rich physics governing its survival and infection strategies. Self-assembly of bacteria at the droplet contact line is characterized by order-to-disorder packing transitions with high packing densities and excessive deformations (bacteria deforms nearly twice its original length scales). In contrast, thin-film instability-led hole formation at the center of the droplet engenders spatial packing of bacteria analogous to honeycomb weathering. The varying physical forces acting on bacteria based on their respective spatial location inside the droplet cause an assorted magnitude of physical stress. Self-assembly favors the bacteria at the rim of the droplet, leading to enhanced viability and pathogenesis on the famously known "coffee ring" of the droplet compared to the bacteria present at the center of the droplet residue. Mechanistic insights gained via our study can have far-reaching implications for bacterial infection through droplets, e.g., through open wounds. | microbiology |
10.1101/2022.05.04.490658 | Physics of Self-Assembly and Morpho-Topological Changes of Klebsiella Pneumoniae in Desiccating Sessile Droplets | We have investigated the flow and desiccation-driven self-assembly of Klebsiella Pneumoniae in the naturally evaporating sessile droplets. Klebsiella Pneumoniae exhibits extensive changes in its morphology and forms unique patterns as the droplet dries, revealing hitherto unexplored rich physics governing its survival and infection strategies. Self-assembly of bacteria at the droplet contact line is characterized by order-to-disorder packing transitions with high packing densities and excessive deformations (bacteria deforms nearly twice its original length scales). In contrast, thin-film instability-led hole formation at the center of the droplet engenders spatial packing of bacteria analogous to honeycomb weathering. The varying physical forces acting on bacteria based on their respective spatial location inside the droplet cause an assorted magnitude of physical stress. Self-assembly favors the bacteria at the rim of the droplet, leading to enhanced viability and pathogenesis on the famously known "coffee ring" of the droplet compared to the bacteria present at the center of the droplet residue. Mechanistic insights gained via our study can have far-reaching implications for bacterial infection through droplets, e.g., through open wounds. | microbiology |
10.1101/2022.05.04.490583 | A systematic approach to study protein-substrate specificity enables the identification of Ssh1 substrate range | Many cellular functions are carried out by protein pairs, or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to study transient PPIs systematically are underutilized. In this study we create a novel approach to study stable as well as transient PPIs in yeast. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in Vivo), uses high- throughput pairwise proximity biotin ligation for uncovering PPIs systematically and in vivo. As a proof of concept we study the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally this demonstrates how CEl-lctiv can provide direct information on substrate specificity even for highly homologous proteins. | cell biology |
10.1101/2022.05.04.490630 | High-throughput single-cell sequencing for retroviral reservoir characterization | During the course of infection, human immunodeficiency virus (HIV) maintains a stably integrated reservoir of replication-competent proviruses within the host genome that are unaffected by antiretroviral therapy. Curative advancements rely heavily on targeting the reservoir, though determinants of its evolutionary origins remain ill-supported through current strategies and are often limited by sample variety. Here, we describe a single-cell deoxyribonucleic acid sequencing (scDNA-seq) method, optimized for sequencing of proviral and host DNA from a treatment-interrupted HIV animal model. We report its benefits for improving viral reservoir resolution to support critical evolutionary events otherwise considered unreliable using traditional viral envelope gene signal alone, as well as comparative advantages to existing near-full-length genome sequencing methods. Given the variety of proviral characteristics that may influence viral rebound, scDNA-seq holds immense value in its ability to streamline many of the present-day applications available in viral reservoir studies, such as integration status and putative replication competency. | genomics |
10.1101/2022.05.04.490682 | Cirrhosis and Inflammation Regulates CYP3A4 Mediated Chemoresistance in Vascularized Hepatocellular Carcinoma-on-a-chip | Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient-specific treatment strategies. In this work, we created novel three-dimensional vascularized HCC-on-a-chips (HCCoC), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness. HCC inflammation was controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450-3A4 (CYP3A4) was investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allowed for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). We showed that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increase vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, we show that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy-associated endothelial porosity increase. The HCCoCs were shown to have utility in uncovering the impact of the tumor microenvironment (TME) during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies.
Statement of SignificanceRegulation of drug metabolism during the cancer progression of hepatocellular carcinoma (HCC) can be influential to develop personalized treatment strategies. We created novel vascularized hepatocellular carcinoma-chip (HCCoC) composed of tunable collagen and four main liver-specific cell lines to be used as a preclinical tool. In this model, we found cancer evolution states such as inflammation and cirrhosis increases vascular permeability progressively as a result of increased inflammatory cytokines. Furthermore, delivery of doxorubicin only with embolization improved treatment efficacy by decreasing CYP3A4 activity, which can modulate treatment outcome. Overall, we found different disease states can be influential on CYP3A4, thus its targeting can improve HCC treatment outcome. | bioengineering |
10.1101/2022.05.04.490627 | Microbial population dynamics decouple nutrient affinity from environmental concentration | How the growth rate of a microbial population depends on the availability of chemical nutrients and other resources in the environment is a fundamental question in microbiology. Models of this dependence, such as the well-known Monod model, are characterized by a threshold concentration (or affinity) of the resource, above which the population can grow near its maximum rate. Even though this concept is a core element of microbiological and ecological modeling, there is little empirical and theoretical evidence for the evolved diversity of these thresholds. In particular, does resource scarcity drive populations to evolve commensurately lower thresholds? To address this question, we perform the largest-to-date meta-analysis of resource threshold data across a wide range of organisms and resources, substantially expanding previous surveys. We find that the thresholds vary across orders of magnitude, even for the same organism and resource. To explain this variation, we develop an evolutionary model to show that demographic fluctuations (genetic drift) can constrain the adaptation of resource thresholds. We find that this effect fundamentally differs depending on the type of population dynamics: populations undergoing periodic bottlenecks of fixed size will adapt their thresholds in proportion to the environmental resource concentration, but populations undergoing periodic dilutions of fixed size will evolve thresholds that are largely decoupled from the environmental concentration. Our model not only provides testable predictions for laboratory evolution experiments, but it also reveals how an evolved resource threshold may not reflect the organisms environment. In particular, this explains how organisms in nutrient-rich environments can still evolve facultative growth at low resource concentrations. Altogether our results demonstrate the critical role of population dynamics in shaping fundamental ecological traits. | microbiology |
10.1101/2022.05.04.490663 | Spt6 directly interacts with Cdc73 and is required for Paf1C recruitment to active genes | Paf1C is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C recruitment to Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 remain to be elucidated. Using an in vivo site-specific protein cross-linking strategy, we identified direct interactions between Cdc73 and two components of the elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Through in vitro protein binding assays and crosslinking/mass spectrometry, we show that Cdc73 and Spt6 can interact in the absence of additional factors and propose a binding interface. Rapid depletion of Spt6 dissociated Paf1 from chromatin and altered patterns of Paf1C-dependent histone modifications genome-wide. These results reveal previously unrecognized interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to Paf1C recruitment to active genes in Saccharomyces cerevisiae. | molecular biology |
10.1101/2022.05.04.490577 | Best practice recommendations for sample preservation in metabarcoding studies: a case study on diatom environmental samples | The development of DNA metabarcoding and High-Throughput Sequencing for diatoms is nowadays offering an interesting approach to assess their communities in freshwater and marine ecosystems. In the context of the implementation of these genomic methods to environmental monitoring, protocol constraints are moving from scientific to operational applications, requiring operational guidelines and standards. In particular, the first steps of the diatom metabarcoding process, which consist of sampling and storage, have been addressed in various ways in scientific and pilot studies.
The objective of this study was to compare three currently applied preservation protocols through different storage durations (ranging from one day to one year) for phytobenthos and phytoplankton samples intended for diatom DNA metabarcoding analysis. The experimental design included four freshwater and two marine samples from sites of diverse ecological characteristics. The impact of the preservation and storage was assessed through diatom metabarcoding endpoints: DNA quality and quantity, diversity and richness, community composition and ecological index values (for freshwater samples). The yield and quality of extracted DNA only decreased for freshwater phytobenthos samples preserved with ethanol. Diatom diversity was not affected and their taxonomic composition predominantly reflects the site origin. Only rare taxa (below 100 reads) differed among methods and durations. Thus, importance of preservation method choice is important for low-density species (rare, invasive, threatened or toxic species). However, for biomonitoring purposes, freshwater ecological index values were not affected whatever the preservation method and duration considered (including ethanol preservation), reflecting the site ecological status.
This study proved that diatom metabarcoding is robust enough to replace or complement the current approach based on morphotaxonomy, paving the way to new possibilities for biomonitoring. Thus, accompanied by operational standards, the method will be ready to be confidently deployed and prescribed in future regulatory monitoring. | molecular biology |
10.1101/2022.05.04.490668 | Elemental content of a host-parasite relationship in the threespine stickleback | Parasite infections are ubiquitous and their effects on hosts may play a role in ecosystem processes. Ecological stoichiometry provides a framework to study linkages between consumers and ecosystem process, but the stoichiometric traits of host-parasite associations are rarely quantified. Specifically, whether parasites elemental ratios closely resemble those of their host or if infection is related to host stoichiometry remains less known. To answer such questions, we measured the elemental content (%C, %N, and %P) and ratios (C:N, C:P, and N:P) of parasitized and unparasitized Gasterosteus aculeatus (three-spined stickleback) and their cestode parasite, Schistocephalus solidus. Host and parasite elemental content were distinct from each other, and parasites were generally higher in %C and lower in %N and %P. Parasite infections were related to some elemental ratios, specifically C:N, with more intense parasite infections corresponding to hosts with lower C:N ratio. Parasite stoichiometry was independent of their host and there was no relationship between host and parasite stoichiometry. Instead, parasite body mass and parasite density were important drivers of parasite stoichiometry where larger parasites had lower %C, %N, and %P. Overall, these potential effects of parasite infections on host stoichiometry along with parasites distinct elemental compositions suggest parasites may further contribute to how hosts store and cycle nutrients. | ecology |
10.1101/2022.05.04.490652 | Global genomic epidemiology, resistome, virulome and plasmidome of the Extraintestinal Pathogenic Escherichia coli (ExPEC) ST38 lineage | Most of the extraintestinal human infections worldwide are determined by some specific Extraintestinal Pathogenic Escherichia coli (ExPEC) lineages, which also present a zoonotic character. One of these lineages belongs to ST38, which is considered a uropathogenic/enteroaggregative E. coli hybrid and a high-risk globally disseminated ExPEC. To get insights on the aspects of ST38 epidemiology and evolution as a multidrug resistant and pathogenic lineage, this study performed a global comparative genomic analysis on ST38 genomes. A total of 376 genomes recovered from environments, humans, livestock, wild and domestic animals in all continents throughout three decades were analyzed considering the ST38 phylogenomics, resistome, plasmidome, and virulome. In general, our analyses revealed that, independently of host sources and geographic origin, these genomes were distributed in two clusters comprising clonal and non-clonal genomes. Moreover, the ST38 accessory genome was not strictly associated with clusters and sub-clusters. Interestingly, the High Pathogenicity Island (HPI) prevailed in the major cluster (cluster 2), which comprised most of the genomes from human origin recovered worldwide (2000 to 2020). In addition, the ExPEC ST38 harbors a huge and diverse plasmidome that might contribute to the evolution of this lineage, reflecting in ST38 ability to live in a diversity of niches as a commensal or pathogenic organism. | genomics |
10.1101/2022.05.04.490689 | Thermal adaptation and fatty acid profiles of bone marrow and muscles in mammals: implications of a study of caribou (Rangifer tarandus caribou) | Mammals have evolved several physiological mechanisms to cope with changes in ambient temperature. Particularly critical among them is the process of keeping cells in a fluid phase to prevent metabolic dysfunction. In this paper, we examine variation in the fatty acid composition of bone marrow and muscle tissues in the cold-adapted caribou (Rangifer tarandus caribou) to determine whether there are systematic differences in fatty acid profiles between anatomical regions that could potentially be explained by thermal adaptation. Our results indicate that the bone marrow and muscle tissues from the appendicular skeleton are more unsaturated than the same tissues in the axial skeleton, a finding that is consistent with physiological adaptation of the appendicular regions to thermal challenges. Because mechanisms of thermal adaptation appear to be widely shared among terrestrial mammals, we suggest that the same patterns may prevail in other species, possibly including humans. | zoology |
10.1101/2022.05.04.490688 | A potent and selective inhibitor for the modulation of MAGL activity in the neurovasculature | Chronic inflammation and blood-brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimers disease and Parkinsons disease. Major drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE2 and PGD2. In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood-brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood-brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood-brain barrier dysfunction caused by inflammatory insults. | neuroscience |
10.1101/2022.05.04.490641 | The Tryptophan Metabolizing Enzyme Indoleamine 2,3-Dioxygenase 1 Regulates Polycystic Kidney Disease Progression | Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability exceeding genic effects. Dysregulated metabolism and immune cell function are key disease modulators. The tryptophan metabolites, kynurenines, produced through IDO1, are known immunomodulators. Here, we study the role of tryptophan metabolism in PKD using an orthologous disease model (C57Bl/6J Pkd1RC/RC). We found elevated kynurenine and IDO1 levels in Pkd1RC/RC kidneys versus wildtype. Further, IDO1 levels were increased in ADPKD cell lines and patient cyst cells. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity as measured by %kidney weight/body weight and cystic index. Consistent with a immunomodulatory role of kynurenines, Pkd1RC/RC;Ido1-/- mice presented with significant changes in the cystic immune microenvironment (CME) versus controls. Of note, kidney macrophage numbers decreased and CD8+ T cell numbers increased, both known PKD modulators. Also, pharmacological IDO1 inhibition using a tryptophan analog in Pkd1RC/RC animals resulted in less severe PKD versus controls with similar changes in the CME as in the genetic model. Together, our data suggest that tryptophan metabolism is dysregulated in ADPKD and that its inhibition results in changes to the CME and slows disease progression, making IDO1 a novel therapeutic target for ADPKD. | molecular biology |
10.1101/2022.05.04.490592 | The H2A.Z.1/PWWP2A/NuRD-associated protein HMG20A controls early head and heart developmental transcription programs | Abstract/SummarySpecialized chromatin-binding proteins are required for DNA-based processes during development. We recently established PWWP2A as direct histone variant H2A.Z interactor involved in mitosis and cranial-facial development. Here, we identify the H2A.Z/PWWP2A-associated protein HMG20A as part of several chromatin-modifying complexes including NuRD, and show that it localizes to genomic regulatory regions. Hmg20a depletion causes severe head and heart developmental defects in Xenopus laevis. Our data indicate that craniofacial malformations are caused by defects in neural crest cell (NCC) migration and cartilage formation. These developmental defects are pheno-copied in HMG20A-depleted mESCs, which show inefficient differentiation into NCCs and cardiomyocytes (CMs). Accordingly, loss of HMG20A caused striking deregulation of transcription programs involved in epithelial- mesenchymal transition (EMT) and cardiac differentiation, thereby providing insights into the regulatory circuits controlled by HMG20A. Collectively, our findings implicate HMG20A as part of the H2A.Z/PWWP2A/NuRD-axis and reveal it as a key modulator of the intricate developmental transcription programs that guide NCC and cardiomyocyte differentiation. | molecular biology |
10.1101/2022.05.04.490255 | Expression and functional evaluation of recombinant human ABO blood group antigen cleaving glycoside hydrolases α-N-acetylgalactosaminidase, α-galactosidase, and endo-β-galactosidase produced in Escherichia coli | Blood transfusions are an integral component of healthcare; however, availability of viable blood is limited by patient-donor blood type specificity, which contributes to seasonal shortages as well as shortages worldwide, especially in developing countries, and during pandemics or natural disasters. Attempts to increase blood supply with commercial incentives have raised ethical concerns, and current proposed artificial blood substitutes are unable to fully replicate the function of native red blood cells (RBCs). In this study, we explore the potential strategy of alleviating blood shortages through enzymatic conversion of A, B, and AB blood types to blood type O. In theory, this process eliminates ABO patient-donor incompatibility, which increases the supply of universal donor blood. Three glycoside hydrolases, -N-acetylgalactosaminidase, -galactosidase, and endo-{beta}-galactosidase, were selected to act as molecular scissors to cleave terminal residues on A and B RBC surface antigens and catalyze the conversion process. These enzymes were recombinantly expressed in BL21(DE3) Escherichia coli and purified through nickel ion affinity chromatography. A combination of colorimetric substrate assays, thin-layer chromatography, and mass spectroscopy were utilized to evaluate enzyme functionality. Enzyme efficiency was modeled using Michaelis-Menten kinetics. Partial enzymatic A-to-O blood type conversion on porcine red blood cells was observed with slide agglutination tests. Results confirm recombinant enzyme-mediated blood type conversion as a potential strategy for alleviating blood shortages. | biochemistry |
10.1101/2022.05.04.490255 | Expression and functional evaluation of recombinant human ABO blood group antigen cleaving glycoside hydrolases α-N-acetylgalactosaminidase, α-galactosidase, and endo-β-galactosidase produced in Escherichia coli | Blood transfusions are an integral component of healthcare; however, availability of viable blood is limited by patient-donor blood type specificity, which contributes to seasonal shortages as well as shortages worldwide, especially in developing countries, and during pandemics or natural disasters. Attempts to increase blood supply with commercial incentives have raised ethical concerns, and current proposed artificial blood substitutes are unable to fully replicate the function of native red blood cells (RBCs). In this study, we explore the potential strategy of alleviating blood shortages through enzymatic conversion of A, B, and AB blood types to blood type O. In theory, this process eliminates ABO patient-donor incompatibility, which increases the supply of universal donor blood. Three glycoside hydrolases, -N-acetylgalactosaminidase, -galactosidase, and endo-{beta}-galactosidase, were selected to act as molecular scissors to cleave terminal residues on A and B RBC surface antigens and catalyze the conversion process. These enzymes were recombinantly expressed in BL21(DE3) Escherichia coli and purified through nickel ion affinity chromatography. A combination of colorimetric substrate assays, thin-layer chromatography, and mass spectroscopy were utilized to evaluate enzyme functionality. Enzyme efficiency was modeled using Michaelis-Menten kinetics. Partial enzymatic A-to-O blood type conversion on porcine red blood cells was observed with slide agglutination tests. Results confirm recombinant enzyme-mediated blood type conversion as a potential strategy for alleviating blood shortages. | biochemistry |
10.1101/2022.05.04.490414 | AptaMat: a matrix-based algorithm to compare single-stranded oligonucleotides secondary structures | Comparing single-stranded nucleic acids (ssNAs) secondary structures is fundamental when investigating their function and evolution and predicting the effect of mutations on the ssNAs structures. Many comparison metrics exist, although they are either too elaborate or not enough sensitive to distinguish close ssNAs structures.
In this context, we developed AptaMat, a simple and sensitive algorithm for ssNAs secondary structures comparison based on matrices representing the ssNAs secondary structures and a metric built upon the Manhattan distance in the plane. We applied AptaMat to several examples and compared the results to those obtained by the most frequently used metrics, namely the Hamming distance and the RNAdistance, and by a recently developed image-based approach. We showed that AptaMat is able to discriminate between similar sequences, outperforming all the other here considered metrics. | bioinformatics |
10.1101/2022.05.04.490567 | Identification of neoantigens in esophageal adenocarcinoma | Esophageal adenocarcinoma (EAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumor mutations, presented to cytotoxic T-cells by human leukocyte antigen molecules (HLA). Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumors from a cohort of seven patients with EAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification. | cancer biology |
10.1101/2022.05.04.490602 | High-resolution mass spectrometry reveals environmentally relevant uptake, elimination, and metabolic alterations following early embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish | Dioxin and dioxin-like compounds are ubiquitous environmental contaminants that induce toxicity by binding to the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The zebrafish model has been used to define the developmental toxicity observed following exposure to exogenous AHR ligands such as the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD). While the model has successfully identified cellular targets of TCDD and molecular mechanisms mediating TCDD-induced phenotypes, fundamental information such as the body burden produced by standard exposure paradigms is still unknown. We performed targeted gas chromatography (GC) high-resolution mass spectrometry (HRMS) in tandem with non-targeted liquid chromatography (LC) HRMS to quantify TCDD uptake, model the elimination dynamics of TCDD, and determine how TCDD exposure affects the zebrafish metabolome. We found that 10 ppb, 1 ppb, and 50 ppt waterborne exposures during early embryogenesis produced environmentally relevant body burden of TCDD: 38 {+/-} 4.34, 26.6 {+/-} 1.2, and 8.53 {+/-} 0.341 pg/embryo, respectively, at 24 hours post fertilization. In addition, we discovered that TCDD exposure was associated with the dysregulation of several metabolic pathways that are critical for brain development and function including glutamate metabolism, chondroitin sulfate biosynthesis, and tyrosine metabolism pathways. Together, these data demonstrate that existing exposure paradigms produce environmentally relevant body burdens of TCDD in zebrafish and provide insight into the biochemical pathways impacted by toxicant-induced AHR activation.
HIGHLIGHTSO_LIHistorical TCDD exposure paradigms produce environmentally relevant body burdens in zebrafish embryos.
C_LIO_LITCDD elimination for high doses can be modeled using an exponential regression.
C_LIO_LIExposure to TCDD alters metabolic pathways that are essential for brain development and function.
C_LI
GRAPHICAL ABSTRACT
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[email protected]@1fd19d8org.highwire.dtl.DTLVardef@1abcc85org.highwire.dtl.DTLVardef@38d3_HPS_FORMAT_FIGEXP M_FIG C_FIG | pharmacology and toxicology |
10.1101/2022.05.04.490602 | High-resolution mass spectrometry reveals environmentally relevant uptake, elimination, and metabolic alterations following early embryonic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish | Dioxin and dioxin-like compounds are ubiquitous environmental contaminants that induce toxicity by binding to the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The zebrafish model has been used to define the developmental toxicity observed following exposure to exogenous AHR ligands such as the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD). While the model has successfully identified cellular targets of TCDD and molecular mechanisms mediating TCDD-induced phenotypes, fundamental information such as the body burden produced by standard exposure paradigms is still unknown. We performed targeted gas chromatography (GC) high-resolution mass spectrometry (HRMS) in tandem with non-targeted liquid chromatography (LC) HRMS to quantify TCDD uptake, model the elimination dynamics of TCDD, and determine how TCDD exposure affects the zebrafish metabolome. We found that 10 ppb, 1 ppb, and 50 ppt waterborne exposures during early embryogenesis produced environmentally relevant body burden of TCDD: 38 {+/-} 4.34, 26.6 {+/-} 1.2, and 8.53 {+/-} 0.341 pg/embryo, respectively, at 24 hours post fertilization. In addition, we discovered that TCDD exposure was associated with the dysregulation of several metabolic pathways that are critical for brain development and function including glutamate metabolism, chondroitin sulfate biosynthesis, and tyrosine metabolism pathways. Together, these data demonstrate that existing exposure paradigms produce environmentally relevant body burdens of TCDD in zebrafish and provide insight into the biochemical pathways impacted by toxicant-induced AHR activation.
HIGHLIGHTSO_LIHistorical TCDD exposure paradigms produce environmentally relevant body burdens in zebrafish embryos.
C_LIO_LITCDD elimination for high doses can be modeled using an exponential regression.
C_LIO_LIExposure to TCDD alters metabolic pathways that are essential for brain development and function.
C_LI
GRAPHICAL ABSTRACT
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[email protected]@1fd19d8org.highwire.dtl.DTLVardef@1abcc85org.highwire.dtl.DTLVardef@38d3_HPS_FORMAT_FIGEXP M_FIG C_FIG | pharmacology and toxicology |
10.1101/2022.05.04.490615 | Visualizing cell-cell interactions between immune effector and tumor cells induced by antibodies using a proximity-dependent biosensor system | Therapeutic antibodies can engage innate (NK) and adaptive (T) immune cells to eliminate pathogenic or malignant cells. Activated NK or T cells exert cytotoxic effects dependent upon direct cell-cell contact and subsequent formation of immunological synapses (IS) with target cells. Therefore, understanding antibody-mediated cell-cell interactions is crucial to optimizing antibody pharmacology and efficacy across all therapeutic areas. To date, most investigations around antibody-mediated cell-cell interactions have primarily focused on molecular-scale interactions. In this study, we developed a biosensor system to evaluate direct cell-cell contact and interactions and the formation of IS at the cell population level. In this biosensor system, two structural complementary luciferase units (SmBit and LgBit) were respectively expressed on immune effector and target cell membranes with optimized spacer lengths. Upon cell-cell contact, two subunits come together to form active NanoLuc, generating a luminescent signal for longitudinal monitoring. The selectivity of the system were optimized by adjusting the spacer lengths to assure the signal is from stable cell-cell interactions with minimal interference from nonspecific and transient intercellular contact. This system was then applied to longitudinally quantify cell-cell interactions between NK and target cells induced by an anti-CD20 antibody (rituximab) and between T and target cells induced by a bispecific (anti-CD3/CD19) T cell engaging antibody (blinatumomab) in a three-dimensional cell culture system. This biosensor system shows promise for monitoring cell-cell interactions in physiologically relevant environments and provides insights into the environmental factors that impede cell-cell interaction and antibody efficacy. | pharmacology and toxicology |
10.1101/2022.05.04.490403 | A computational toolbox to investigate the metabolic potential and resource allocation in fission yeast | The fission yeast Schizosaccharomyces pombe is a popular eukaryal model organism for cell division and cell cycle studies. With this extensive knowledge of its cell and molecular biology, S. pombe also holds promise for use in metabolism research and industrial applications. However, unlike the bakers yeast Saccharomyces cerevisiae, a major workhorse in these areas, cell physiology and metabolism of S. pombe remain less explored. One way to advance understanding of organism-specific metabolism is construction of computational models and their use for hypothesis testing. To this end, we leverage existing knowledge of S. cerevisiae to generate a manually-curated high-quality reconstruction of S. pombes metabolic network, including a proteome-constrained version of the model. Using these models, we gain insights into the energy demands for growth, as well as ribosome kinetics in S. pombe. Furthermore, we predict proteome composition and identify growth-limiting constraints that determine optimal metabolic strategies under different glucose availability regimes, and reproduce experimentally determined metabolic profiles. Notably, we find similarities in metabolic and proteome predictions of S. pombe with S. cerevisiae, which indicate that similar cellular resource constraints operate to dictate metabolic organization. With these use cases, we show, on the one hand, how these models provide an efficient means to transfer metabolic knowledge from a well-studied to a lesser-studied organism, and on the other, how they can successfully be used to explore the metabolic behaviour and the role of resource allocation in driving different strategies in fission yeast. | systems biology |
10.1101/2022.05.04.490603 | Morphological evolution and diversity of pectoral fin skeletons in teleosts | The Teleostei class has the most species of the fishes. Members of this group have paired rostral appendages and pectoral fins, enabling refined movements in the water. Although teleosts live in a diverse set of environments, the skeletal pattern of pectoral fins in teleosts is considered to show little morphological variability. Here, in order to elucidate variations in pectoral fin skeletons and to identify their evolutionary processes, we compared the pectoral fin skeletons from 27 species of teleosts. We identified several variations and a diversity of pectoral fin skeletal patterns within some teleost groups. Taken together with previous reports on teleost skeletons, our findings reveal that in the course of teleost evolution, there are a mixture of conserved and non- conserved components in the pectoral fin skeletons of teleosts, and that teleosts may have experienced the variation and conservation of the number and shape of the proximal radials, the loss of the mesocoracoid, and the change in the distal radial-fin ray relationship. | zoology |
10.1101/2022.05.04.490528 | Ribotoxic collisions on CAG expansions disrupt proteostasis and stress responses in Huntington's Disease | Huntingtons disease (HD) is a devastating neurodegenerative disorder caused by CAG trinucleotide repeat expansions encoding a polyglutamine (polyQ) tract in the Huntingtin (HTT) gene1. Although mutant HTT (mHTT) protein tends to aggregate, the exact causes of neurotoxicity in HD remain unclear2. Here we show that altered elongation kinetics on CAG expansions cause ribosome collisions that trigger ribotoxicity, proteotoxicity and maladaptive stress responses. CAG expansions cause an elongation rate conflict during HTT translation, when ribosomes rapidly decoding the optimal polyQ encounter a flanking slowly-decoded polyproline tract. The ensuing ribosome collisions lead to premature termination and release of aggregation-prone mHTT fragments. Due to the presence of a stress-responsive upstream open reading frame (uORF), HTT translation and aggregation are limited under normal conditions but enhanced under stress, seeding a vicious cycle of dysfunction. mHTT further exacerbates ribotoxicity by progressively sequestering eIF5A, a key regulator of translation elongation, polyamine metabolism and stress responses. eIF5A depletion in HD cells leads to widespread ribosome pausing on eIF5A-dependent sites, impaired cotranslational proteostasis, disrupted polyamine metabolism and maladaptive stress responses. Importantly, drugs that reduce translation initiation attenuate ribosome collisions and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD. | neuroscience |
10.1101/2022.05.04.490484 | Behavioral and brain responses to verbal stimuli reveal transient periods of cognitive integration of external world in all sleep stages | Sleep has long been considered as a state of disconnection from the environment, with absent reactivity to external stimuli. Here, we questioned this sleep disconnection dogma by directly investigating behavioral responsiveness in 49 napping subjects (27 with narcolepsy and 22 healthy volunteers) engaged in a lexical decision task. Participants were instructed to frown or smile depending on the stimulus type (words vs pseudo-words). We found accurate behavioral responses, visible via contractions of the corrugator or zygomatic muscles, in all sleep stages in both groups (except slow-wave sleep for healthy volunteers). Stimuli presented during states with high (vs. low) values of neural markers indexing rich cognitive states more often yielded responses. Our findings suggest that transient windows of reactivity to external stimuli exist in all sleep stages, even in healthy individuals. Such windows of reactivity could be used to probe sleep-related mental and cognitive processes in real-time across all sleep stages. | neuroscience |
10.1101/2022.05.04.490030 | Perceptual cycles travel across retinotopic space | Visual perception waxes and wanes periodically as a function of the phase of low-frequency brain oscillations (theta, 4-7 Hz; alpha, 8-13 Hz) [1-9]. Perceptual cycles are defined as the corresponding periodic modulation of perceptual performance (review [10, 11]). Here, using psychophysics, we tested the hypothesis that brain oscillations travel across the visual cortex, leading to predictable perceptual consequences across the visual field, i.e., perceptual cycles travel across the retinotopic visual space. An oscillating disk (inducer) was presented in the periphery of the visual field to induce brain oscillations at low frequencies (4, 6, 8 or 10 Hz) at a specific retinotopic cortical location. Target stimuli at threshold (50% detection) were displayed at random times during the periodic disk stimulation, at one of three possible distances from the disk. Electroencephalography (EEG) was recorded while participants performed a detection task. EEG analyses showed that the periodic stimulation produced a complex brain oscillation composed of the induced frequency and its first harmonic likely due to the overlap of the periodic response and the neural response to individual stimuli. This complex oscillation, which originated from a precise retinotopic position, modulated detection performance periodically at each target position and at each frequency. Critically, the optimal behavioral phase, i.e., of highest performance, of the 8 Hz and 10 Hz oscillations (alpha range) consistently shifted across target distance to the inducer. Together, the results demonstrate that alpha-induced perceptual cycles traveled across the retinotopic space in human observers at a propagation speed between 0.2 and 0.4 m/s. | neuroscience |
10.1101/2022.05.04.490638 | Task prioritization modulates low frequency EEG dynamics reflecting proactive cognitive control | Most neuroscientific studies investigating mental effort apply unspecific effort allocation paradigms. In contrast, the present EEG study targets specific effort allocation during task prioritization.
Twenty-eight participants performed a cued number classification task during the retention interval of a working memory task including retrospective cues. One of two possible number classifications was done per trial. Each trial started with a cue indicating which of the two tasks would be more important in the upcoming trial. Subjects were told to engage in both tasks, but to concentrate on the important one. Feedback given at the end of each trial was calculated based on task performance, with scores obtained from the relevant task being tripled.
Participants performed significantly better in either task when it was important compared to when not.Task prioritization modulates theta, alpha and beta oscillations, predominantly during task preparation. Multivariate pattern analysis revealed that the exact type of the two possible number classifications was decodable, however, decoding accuracy did not depend on task importance. Hemispheric alpha power asymmetries indicating attentional orienting between working memory representations also did not depend on task importance. The findings suggest that task prioritization primarily affects proactive cognitive control on a superordinate level. | neuroscience |
10.1101/2022.05.04.490634 | Expression of GDNF Receptors Within Nucleus Ambiguus During Rat Development | ObjectiveUpregulation of GDNF and its receptors is observed during laryngeal reinnervation after nerve injury. In contrast, little is known regarding the expression of GDNF receptors in the formation of the nucleus ambiguus (NA) and its innervation of the larynx during embryogenesis. Differences may suggest therapeutic targets after nerve injury.
Study DesignLaboratory experiment.
MethodsRat brainstems at E14, E16, E18, E20, adult (4 animals/timepoint) were sectioned and stained for GDNF receptors: GFR-1, GFR-2, GFR-3, and Ret. Islet1 and ChAT were used as markers for motoneuron cell bodies. Sections were observed using Zeiss Axio Imager M2 Microscope and quantified using Image J.
ResultsExpression of all four GDNF receptors was identified within the nucleus ambiguus, as well as hypoglossal and facial nuclei of the adult rat brainstem. During rat development, GFR-3 and Ret exhibited upregulation within the nucleus ambiguus at E14 whereas GFR-1 began showing upregulation at E20. GFR-2 exhibited no upregulation at embryonic timepoints. Conclusion: Upregulation of the GDNF receptors within the nucleus ambiguus occur after laryngeal muscles innervation during development and may associated with maturation and maintenance of the neuromuscular synapses of the larynx before and after birth. No differences among ambiguus, hypoglossal, and facial nuclei was observed. | neuroscience |
10.1101/2022.05.04.490596 | Plasma miRNA-214 is a predictive candidate biomarker of progression speed in patients with ALS | This study was designed to develop and validate a reliable biomarker to predict the progression speed reflecting immune function of amyotrophic lateral sclerosis (ALS). After establishing the induced microglia model (iMGs) derived from peripheral blood monocytes, comparative studies to find factors related to phagocytic differences between iMGs of patients with rapidly progressive ALS [ALS(R)-iMGs, n = 15] and those of patients with slowly progressive ALS [ALS(S)-iMGs, n = 14] were conducted in the discovery cohort. To validate discovered candidate and whether it could be used as a reliable biomarker predicting the progression speed of ALS, we recruited 132 patients with ALS and 30 age-matched healthy controls as the validation cohort. ALS(R)-iMGs showed impaired phagocytic function. Transcriptomic analysis revealed that the perturbed phagocytosis in ALS(R)-iMGs was related to the decreased expression of NCKAP1 (NCK-associated protein 1) and NCKAP1 overexpression rescued the impaired phagocytic function. miRNA-214-3p targeting NCKAP1 in ALS-iMGs was correlated with progression speed in the discovery cohort. The validation cohort revealed that plasma miRNA-214-3p levels were significantly increased in ALS patients (p < 0.0001, AUC = 0.839), correlated with disease progression speed (p = 0.0005), and distinguished the rapidly progressive subgroup (Q1) from the slowly progressive (Q4, p = 0.029), respectively. Plasma miRNA-214-3p can predict the progression speed in ALS. Plasma miRNA-214-3p could be used as a simple and easily accessible biomarker for predicting the future progression speed related to phagocytic dysfunction in ALS patients. | neuroscience |
10.1101/2022.05.04.489500 | A pro-oxidant combination of resveratrol and copper down-regulates multiple biological hallmarks of ageing and neurodegeneration | Several hundred billion to a trillion cells die in the body every day, and cell-free chromatin particles (cfChPs) that are released from them enter into the extracellular compartments of the body, including into the circulation. We have earlier reported that cfChPs can readily enter into healthy cells to damage their DNA, activate apoptotic pathways and induce inflammatory cytokines. We hypothesized that repeated lifelong assault on healthy cells by cfChPs is the underlying cause of ageing, and that the ageing process could be retarded by deactivating cfChPs. The latter can be effected by oxygen radicals that are generated upon admixing the nutraceuticals resveratrol (R) and copper (Cu). Using confocal microscopy and antibodies against DNA and histone we detected copious presence of extra-cellular cfChPs in brain of ageing mice, and observed that these were deactivated / eradicated following prolong oral administration of small quantities of R-Cu. Deactivation / eradication of cfChPs was associated with down-regulation of several biological hallmarks of ageing in brain cells which included reduction in: 1) telomere attrition, 2) amyloid deposition, 3) DNA damage, 4) apoptosis, 5) inflammation, 6) senescence, 7) aneuploidy and 8) mitochondrial dysfunction. At a systemic level, R- Cu treatment led to significant reduction in blood levels of glucose, cholesterol and C-reactive protein. These results suggest that cfChPs may be global instigators of ageing and neurodegeneration, and that therapeutic use of R-Cu may help to retard the process of ageing. | neuroscience |
10.1101/2022.05.04.490570 | Retinoic acid-gated BDNF synthesis in neuronal dendrites drives presynaptic homeostatic plasticity | Homeostatic synaptic plasticity is a non-Hebbian synaptic mechanism that adjusts synaptic strength to maintain network stability while achieving optimal information processing. Among the molecular mediators shown to regulate this form of plasticity, synaptic signaling through retinoic acid (RA) and its receptor, RAR, has been shown to be critically involved in the homeostatic adjustment of synaptic transmission in both hippocampus and sensory cortices. In this study, we explore the molecular mechanism through which postsynaptic RA and RAR regulates presynaptic neurotransmitter release during prolonged synaptic inactivity at excitatory synapses. We show that RAR binds to a subset of dendritically sorted brain-derived neurotrophic factor (BDNF) mRNA splice isoforms and represses their translation. The RA-mediated translational de-repression of postsynaptic BDNF results in the retrograde activation of presynaptic Tropomyosin receptor kinase B (TrkB) receptors, facilitating presynaptic homeostatic compensation through enhanced presynaptic release. Together, our study illustrates a RA-mediated retrograde synaptic signaling pathway through which postsynaptic protein synthesis during synaptic inactivity drives compensatory changes at presynaptic site. | neuroscience |
10.1101/2022.05.04.490593 | Multivariate Analysis of PET Pharmacokinetic Parameters | PurposeIn positron emission tomography (PET) quantification, multiple pharmacokinetic parameters are typically estimated from each time activity curve. Conventionally, all but the parameter of interest are discarded before performing subsequent statistical analysis. However, we assert that these discarded parameters also contain relevant information which can be exploited to improve the precision and power of statistical analyses on the parameter of interest. Properly taking this into account can thereby draw more informative conclusions without collecting more data.
MethodsBy applying a hierarchical multifactor multivariate Bayesian approach, all estimated parameters from all regions can be analysed at once. We refer to this method as PuMBA (Parameters undergoing Multivariate Bayesian Analysis). We simulated patientcontrol studies with different radioligands, varying sample sizes and measurement error to explore its performance, comparing the precision, statistical power, false positive rate and bias of estimated group differences relative to univariate analysis methods.
ResultsWe show that PuMBA improves the statistical power for all examined applications relative to univariate methods without increasing the false positive rate. PuMBA improves the precision of effect size estimation, and reduces the variation of these estimates between simulated samples. Furthermore, we show that PuMBA yields performance improvements even in the presence of substantial measurement error. Remarkably, owing to its ability to leverage information shared between pharmacokinetic parameters, PuMBA even shows greater power than conventional univariate analysis of the true binding values from which the parameters were simulated. Across all applications, PuMBA exhibited a small degree of bias in the estimated outcomes, however this was small relative to the variation in estimated outcomes between simulated datasets.
ConclusionPuMBA improves the precision and power of statistical analysis of PET data without requiring the collection of additional measurements. This makes it possible to study new research questions in both new and previously collected data. PuMBA therefore holds great promise for the field of PET imaging. | neuroscience |
10.1101/2022.05.04.490582 | KIF21B binds Myosin Va for Spine Entry and regulates Actin Dynamics to control Homeostatic Synaptic Downscaling | Homeostatic synaptic plasticity adjusts the strength of synapses to restrain neuronal activity within a physiological range. Postsynaptic GKAP controls the bidirectional synaptic scaling of AMPA receptors (AMPARs) however how chronic activity triggers postsynaptic protein remodeling to downscale synaptic transmission is barely understood. Here we report that the microtubule-dependent kinesin motor KIF21B interacts with GKAP and likewise enters dendritic spines in a myosin Va- and activity-dependent manner. We observed that under conditions of chronic activity KIF21B regulates actin dynamics in spines, triggers spine removal of GluA2-containing AMPA receptors, and mediates homeostatic synaptic downscaling of AMPA receptor-mediated mEPSC amplitudes. Our data highlight a myosin-kinesin interaction that enables the entry of the microtubule-dependent motor KIF21B into actin-rich spine compartments. A slow actin turnover rate might be beneficial for efficient protein removal from excitatory synapses, suggesting a functional role of KIF21B in a GKAP- and AMPA receptor-dependent mechanism, underlying homeostatic downscaling of neuronal firing. | neuroscience |
10.1101/2022.05.04.490383 | Chronic lymphocytic leukemia includes a tumor subset resembling memory B cells that develop early and persist | Single-cell RNAseq/VDJseq of tumor cells and normal residual B (NRB) cells from peripheral blood of chronic lymphocytic leukemia (CLL) patients identified three distinct tumor subsets according to phenotype, transcriptome, and immunoglobulin-V-gene (IgV)-mutations. Two major subsets share a typical CLL phenotype but differ in signaling, metabolism and cell cycle control, indicating that the circulating CLL pool is shaped by two states of activity. The third CLL subset shows the phenotype, proliferation capacity and extensive IgV-mutation diversity of normal CD5+ memory B cells. This previously unrecognized CLL tumor subset, which intermingles with NRB cells, was confirmed in 33 IgV-mutated (M-CLL) and IgV-unmutated (U-CLL) cases. Longitudinal IgV-mutation phylogenetics suggest that these NRB-associated CLL cells are generated pathogenetically early, mostly in germinal center reactions, and archive the individual IgV-diversification program, which is conserved throughout CLL course. Our study suggests that diversity is established early in CLL, that each tumor is composed of multiple subclonal expansions, and subclonal evolution can be depicted by IgV-mutation phylogenetics. | immunology |
10.1101/2022.05.04.490595 | The radio-protective effects of (-)-Epigallocatechin-3-gallate (EGCG): regulating macrophage function in radiation-induced intestinal injury. | Accidental and medical radiation exposure may cause radiation-induced intestinal injury (RIII), a catastrophic disease requiring efficient therapies. (-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea, has been shown to have potent biological activity as well as strong anti-inflammation effects. Here, we demonstrate that EGCG treatment not only protects mice against total body irradiation (TBI)-induced toxicity and weight loss but also alleviates whole abdominal irradiation (WAI)-induced intestinal injury. EGCG promotes proliferation and survival of intestinal stem cells, inhibits radiation-induced apoptosis and inflammation. At the same time, EGCG preserves the composition of the gut microbiota in WAI mice. In vitro, we demonstrate that EGCG regulates the release of radiation-induced inflammatory factors by inhibiting inflammatory pathways such as toll-like receptor signaling pathway in peritoneal macrophages. Mechanistically, the radioprotective effect of EGCG was likely attributable to its preservation on macrophages and the colonized gut microbiota composition, thus relieving the intestinal inflammation. EGCG provides a novel strategy to mitigate RIII and improve the prognosis of patients after radiotherapy. | cell biology |
10.1101/2022.05.04.490621 | Inhibiting 5-lipoxygenase prevents skeletal muscle atrophy by targeting organogenesis signaling and insulin-like growth factor-1 | BackgroundSkeletal muscle atrophy can occur in response to numerous factors, such as aging and certain medications, and produces a major socioeconomic burden. At present, there are no approved drugs for treating skeletal muscle atrophy. Arachidonate 5-lipoxygenase (Aox5) is a drug target for a number of diseases. However, pharmacological targeting of Alox5, and its role in skeletal muscle atrophy, is unclear.
MethodsThe potential effects of gene knockdown and pharmacological targeting of Alox5 on skeletal muscle atrophy was investigated using cell-based models, animal models, and human skeletal muscle tissue cultures. Malotilate, a clinically safe drug developed for enhancing liver regeneration and Alox5 inhibitor, was investigated as a repurposing candidate. Mechanism(s) of action in skeletal muscle atrophy were assessed by measuring the expression level or activation status of key regulatory pathways, and validated using gene knockdown and RNA sequencing.
ResultsMyotubes treated with the atrophy-inducing glucocorticoid, dexamethasone, were protected from catabolic responses by treatment with malotilate (+41.29%, P < 0.01). Similar anti-atrophy effects were achieved by gene knockdown of Alox5 (+30.4%, P < 0.05). Malotilate produced anti-atrophy effects without affecting the myogenic differentiation program. In an in vivo model of skeletal muscle atrophy, malotilate treatment enhanced muscle performance (Grip strength: +35.72%, Latency to fall: +553.1%, P < 0.05), increased mass and fiber cross sectional area (Quadriceps: +23.72%, Soleus: +33.3%, P < 0.01), and down-regulated atrogene expression (Atrogin-1: -61.58%, Murf-1: -66.06%, P < 0.01). Similar, beneficial effects of malotilate treatment were observed in an aging muscle, which also showed the preservation of fast twitch fibers (Type 2a: +56.48%, Type 2b: +37.32%, P < 0.01). Leukotrine B4, a product of Alox5 activity with inflammatory and catabolic functions, was found to be elevated in skeletal muscle undergoing atrophy (Quadriceps: +224.4%, P < 0.001). Cellular transcriptome analysis showed that targeting Alox5 upregulated biological processes regulating organogenesis and increased the expression of insulin-like growth factor-1, a key anti-atrophy hormone (+226.5%, P < 0.05). Interestingly, these effects were restricted to the atrophy condition and not observed in normal skeletal muscle cultures with Alox5 inhibition. Human skeletal muscle tissue was also protected from atrophy by pharmacological targeting of Alox5 (+23.68%, P < 0.05).
ConclusionThese results shed new light on novel drug targets and mechanisms underpinning skeletal muscle atrophy. Alox5 is a regulator and drug target for muscle atrophy, and malotilate is an attractive compound for repurposing studies to treat this disease. | cell biology |
10.1101/2022.05.04.490568 | An autonomous mathematical model for the mammalian cell cycle | A mathematical model for the mammalian cell cycle is developed as a system of 13 coupled nonlinear ordinary differential equations. The variables and interactions included in the model are based on detailed consideration of available experimental data. Key features are that the model is autonomous, except for dependence on external growth factors; variables are continuous in time, without instantaneous resets at phase boundaries; cell cycle controllers and completion of tasks associated with cell cycle progression are represented; mechanisms to prevent rereplication are included; and cycle progression is independent of cell size. Eight variables represent cell cycle controllers: Cyclin D1 in complex with Cdk4/6, APCCdh1, SCF{beta}Trcp, Cdc25A, MPF, NUMA, securin-separase complex, and separase. Five variables represent task completion, with four for the status of origins and one for kinetochore attachment. The model predicts distinct behaviors consistent with each main phase of the cell cycle. The response to growth factors shows restriction-point behavior. These results imply that the main features of the mammalian cell cycle can be accounted for in a quantitative mechanistic way based on known interactions among cycle control factors and their coupling to tasks involved in replication of DNA. The model is robust to parameter changes, in that cycling is maintained over at least a five-fold range of each parameter when varied individually. The most sensitive parameters are those associated with the initiation and completion of mitosis. The model is suitable for exploring how extracellular factors affect cell cycle progression, including responses to metabolic conditions and to anti-cancer therapies.
Author SummaryWe created a model, that is, a set of mathematical equations, to represent the entire cell cycle in mammals. All terms in our equations correspond to actual biological mechanisms. We solved the equations and verified that they show similar behavior to real-life cell cycles. We designed our model to cycle only when enough external growth factors stimulate it, as real cells do. Our model helps us better understand how the cell cycle is controlled. One very important aspect of control is how the cell ensures that its DNA is copied only once per cell cycle. If "rereplication" (copying a section of DNA twice in the same cycle) occurs, it can cause harmful DNA damage. We considered several mechanisms that some biologists believe play a role, and found that most could not explain how rare rereplication is. Two mechanisms did work well, and we used them to make two variants of the model, which show similar behavior. The model has many potential applications. The cell cycle is altered in most cancer cells, so understanding how these changes affect control is useful. Many widely-used drugs affect the cell cycle, and our model can be used to study these effects. | cell biology |
10.1101/2022.05.04.490566 | Modelling the spread and mitigation of an emerging vector-borne pathogen:citrus greening in the U.S. | Predictive models, based upon epidemiological principles and fitted to surveillance data, play an increasingly important role in shaping regulatory and operational policies for emerging outbreaks. Data for parameterising these strategically important models are often scarce when rapid actions are required to change the course of an epidemic invading a new region. We provide a flexible toolkit for landscape-scale disease management, which is applicable to a range of emerging pathogens including vector-borne pathogens for both endemic and invading epidemic vectors. We use the toolkit to analyse and predict the spread of Huanglongbing disease or citrus greening in the U.S. We estimate epidemiological parameters using survey data from one region (Texas) and show how to transfer and test parameters to construct predictive spatio-temporal models for another region (California). The models are used to screen effective coordinated and reactive management strategies for different regions. | ecology |
10.1101/2022.05.04.490562 | Effects of microplastics mixed with natural particles on Daphnia magna populations | The toxicity of microplastics on Daphnia magna as key model for freshwater zooplankton is well described. While several studies predict population-level effects based on short-term, individual-level responses, only very few have validated these predictions experimentally. Thus, we exposed D. magna populations to irregular polystyrene microplastics and diatomite as natural particle (both [≤]63 {micro}m) over 50 days. We used mixtures of both particle types at fixed particle concentrations (50,000 mL-1) and recorded the overall population density, the size of the individual animals, and resting egg production. Particle exposure adversely affected the population density and structure and induced resting egg production. The terminal population size was 31-42% lower in exposed compared to control populations. Interestingly, mixtures containing diatomite induced stronger effects than microplastics alone highlighting that natural particles are not per se less toxic than microplastics. Our results demonstrate that an exposure to synthetic and natural particles has negative population-level effects on zooplankton. Understanding the mixture toxicity of microplastics and natural particles is important given that aquatic organisms will experience exposure to both. Just as for chemical pollutants, better knowledge of such joint effects is essential to fully understand the environmental risks of complex particle mixtures.
Environmental ImplicationsWhile microplastics are commonly considered hazardous based on individual-level effects, there is a dearth of information on how they affect populations. Since the latter is key for understanding the environmental impacts of microplastics, we investigated how particle exposures affect the population size and structure of Daphnia magna. In addition, we used mixtures of microplastics and natural particles because neither occurs alone in nature and joint effects can expected in an environmentally realistic scenario. We show that such mixtures adversely affect daphnid populations and highlight that population-level and mixture-toxicity designs are one important step towards more environmental realism in microplastics research.
Graphical Abstract
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[email protected]@552921org.highwire.dtl.DTLVardef@ff461corg.highwire.dtl.DTLVardef@153f479_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDaphnia populations exposed to mixtures of microplastics and diatomite
C_LIO_LIEffects on population density, structure, and resting egg production
C_LIO_LIDiatomite as natural particle was more toxic than microplastics
C_LIO_LIParticle mixtures induce negative population-level effects
C_LIO_LIParticle mixtures represent more realistic exposure scenario
C_LI | ecology |
10.1101/2022.05.04.490581 | Genome mining as a biotechnological tool for the discovery of novel biosynthetic genes in lichens | The ever-increasing demand for novel drugs highlights the need for bioprospecting unexplored taxa for their biosynthetic potential. Lichen-forming fungi (LFF) are a rich source of natural products but their implementation in pharmaceutical industry is limited, mostly because the genes corresponding to a majority of their natural products is unknown. Furthermore, it is not known to what extent these genes encode structurally novel molecules. Advance in next-generation sequencing technologies has expanded the range of organisms that could be exploited for their biosynthetic potential. In this study, we mine the genomes of nine lichen-forming fungal species of the genus Umbilicaria for biosynthetic genes, and categorize the BGCs as "associated product structurally known", and "associated product putatively novel". We found that about 25-30% of the biosynthetic genes are divergent when compared to the global database of BGCs comprising of 1,200,000 characterized biosynthetic genes from planta, bacteria and fungi. Out of 217 total BGCs, 43 were only distantly related to known BGCs, suggesting they encode structurally and functionally unknown natural products. Clusters encoding the putatively novel metabolic diversity comprise PKSs (30), NRPSs (12) and terpenes (1). Our study emphasizes the utility of genomic data in bioprospecting microorganisms for their biosynthetic potential and in advancing the industrial application of unexplored taxa. We highlight the untapped structural metabolic diversity encoded in the lichenized fungal genomes. To the best of our knowledge, this is the first investigation identifying genes coding for NPs with potentially novel therapeutic properties in LFF. | genomics |
10.1101/2022.05.04.490636 | Evaluation of Cognitive Function in the Dog Aging Project: Associations with Baseline Canine Characteristics | Canine Cognitive Dysfunction (CCD) is a neurodegenerative disease in aging dogs. It has been described previously in relatively small cohorts of dogs using multiple different rating scales. This study aimed to use a minimally modified CCD rating scale developed by previous researchers to describe the prevalence of CCD more thoroughly in a large, nationwide cohort of companion dogs participating in the Dog Aging Project (DAP). Associations between various canine characteristics, predicted lifespan quartiles, and CCD were examined using univariable and multivariable logistic regression models and Receiver Operating Curve (ROC) analysis.
When controlling for all other characteristics, the odds of CCD increased 52% with each additional year of age. Among dogs of the same age, health status, breed type, and sterilization status, odds of CCD were 6.47 times higher in dogs who were not active compared to those who were very active. When controlling for age, breed type, activity level, and other comorbidities, dogs with a history of neurological, eye, or ear disorders had higher odds of CCD. Lifespan quartile analysis showed excellent discriminating ability between CCD positive and negative dogs. Weight-based lifespan quartile estimation could therefore serve as a tool to inform CCD screening by veterinarians. | animal behavior and cognition |
10.1101/2022.05.04.490654 | Head-direction tracks internally directed selective attention in visual working memory | We shift our gaze even when we orient attention internally to visual representations in working memory. Here, we show the bodily orienting response associated with internal selective attention is widespread as it also includes the head. In three virtual reality (VR) experiments, participants remembered two visual items. After a working memory delay, a central colour cue indicated which item needed to be reproduced from memory. After the cue, head movements became biased in the direction of the memorised location of the cued memory item - despite there being no items to orient towards in the external environment. The head-direction bias had a distinct temporal profile from the gaze bias. Our findings reveal that directing attention within the spatial layout of visual working memory bears a strong relation to the overt head orienting response we engage when directing attention to sensory information in the external environment. The head-direction bias further demonstrates common neural circuitry is engaged during external and internal orienting of attention. | animal behavior and cognition |
10.1101/2022.05.04.490584 | Plasticity facilitates rapid evolution | Developmental plasticity enables organisms to cope with new environmental challenges. If deploying such plasticity is costly in terms of time or energy, the same adaptive behavior could subsequently evolve through piecemeal genomic reorganisation that replaces the requirement to acquire that adaptation by individual plasticity. Here we report a new dimension to the way in which plasticity can drive evolutionary change leading to ever greater complexity in biological organization. Our model deploys the concept of partially overlapping functional systems. We found that plasticity accelerated dramatically the evolutionary accumulation of adaptive systems in model organisms with relatively low rates of mutation. The effect of plasticity on the evolutionary growth of complexity was even greater when the number of elements needed to construct a functional system was increased. These results suggest that as the difficulty of challenges from the environment become greater, so plasticity exerts an ever more powerful role in meeting those challenges and in opening up new avenues for the subsequent evolution of complex adaptations. | animal behavior and cognition |
10.1101/2022.05.04.490639 | Effects of Body Mass on Leg and Vertical Stiffness in Running Humans | Numerous cross-species comparisons have examined the scaling of gait parameters with respect to body mass (i.e., allometry), but few have done so within humans. This study examined how leg and vertical stiffness, force, displacement, and leg spring angle scaled in 64 healthy adults of varying body masses during slow and fast leg-length-adjusted running speeds. We calculated scaling patterns for stiffness and its components via kinematic and kinetic data using log-log regressions with 95% confidence/highest density intervals. To determine if the chosen statistical method influenced conclusions about scaling patterns, we compared regression results across three statistical methods, ordinary least squares (OLS) regression, linear mixed models (LMM), and Bayesian linear mixed models (BLMM). We also performed sex-specific analyses to determine if each sex revealed similar scaling patterns as the pooled sample. In the pooled sample, all variables scaled according to the isometric expectations, suggesting that different-sized humans move in a similar manner. Sex-specific analyses revealed similar patterns of isometry in all variables, except for vertical stiffness, which displayed slight negative allometry (i.e., lower than expected stiffness) in both sexes at the slow speed and negative allometry in females during fast running. Model choice did not significantly affect results, and scaling patterns were the same regardless of the statistical method employed. | bioengineering |
10.1101/2022.05.04.490563 | FAIRsoft - A practical implementation of FAIR principles for research software | Software plays a crucial and growing role in research. Unfortunately, the computational component in Life Sciences research is challenging to reproduce and verify most of the time. It could be undocumented, opaque, may even contain unknown errors that affect the outcome, or be directly unavailable, and impossible to use by others. These issues are detrimental to the overall quality of scientific research. One step to address this problem is the formulation of principles that research software in the domain should meet to ensure its quality and sustainability, resembling the FAIR (Findable, Accessible, Interoperable and Reusable) Data Principles. Within the ELIXIR infrastructure, OpenEBench aims to be an open platform providing both support for scientific benchmarking and an active observatory of software quality for the universe of Life Sciences research software. We present our initial proposal to instantiate a FAIR-like framework for assessing software quality as the first step toward the implementation of such an observatory in OpenEBench. | bioinformatics |
10.1101/2022.05.04.490585 | A dynamical low-rank approach to solve the chemical master equation for biological reaction networks | Solving the chemical master equation is an indispensable tool in understanding the behavior of biological and chemical systems. In particular, it is increasingly recognized that commonly used ODE models are not able to capture the stochastic nature of many cellular processes. Solving the chemical master equation directly, however, suffers from the curse of dimensionality. That is, both memory and computational effort scale exponentially in the number of species. In this paper we propose a dynamical low-rank approach that enables the simulation of large biological networks. The approach is guided by partitioning the network into biological relevant subsets and thus avoids the use of single species basis functions that are known to give inaccurate results for biological systems. We use the proposed method to gain insight into the nature of asynchronous vs. synchronous updating in Boolean models and successfully simulate a 41 species apoptosis model on a standard desktop workstation. | bioinformatics |
10.1101/2022.05.04.490659 | The effect of motor-induced shaft dynamics on microtubule stability and length | Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depends on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in presence of kinesin-1 molecular motors. Although the average microtubule dynamics can be captured by an effective rescue rate, the dynamics of individual microtubules differs dramatically when rescue occurs only at exchange sites. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network.
SIGNIFICANCEThe microtubule network is essential for vital cellular processes like the organization of intracellular transport and division. Although microtubule assembly occurs at its tips, it has recently been reported that tubulin is exchanged along the microtubule shaft. Tubulin exchange plays an essential role in regulating microtubule dynamics and can be induced by molecular motors. Here, we provide the first systematic study of the impact of shaft dynamics on the regulation of rescue events, where a microtubule switches from shrinking to growing. Our results illustrate how the usage of microtubules as tracks for intracellular transport regulates the microtubule network and thus offers a novel perspective on intracellular organization. | biophysics |
10.1101/2022.05.04.490169 | Human papilloma virus E6 regulates therapy responses in oropharyngeal cancer by repressing the PGC-1α/ERRα axis | Therapy with radiation plus cisplatin kills human papilloma virus-related (HPV+) oropharyngeal squamous cell carcinomas (OPSCCs) by increasing reactive oxygen species beyond cellular antioxidant capacity. To explore why some patients fail these standard treatments, we evaluated whether the variation in HPV oncoprotein levels among HPV+ OPSCCs impacts mitochondrial metabolism, a source of antioxidant capacity. In cell line and patient-derived xenograft models, levels of HPV full-length E6 (fl-E6) inversely correlated with oxidative phosphorylation, antioxidant capacity, and therapy resistance, and fl-E6 was the only HPV oncoprotein to display such correlation. Ectopically expressing fl-E6 in models with low levels reduced mitochondrial mass, depleted antioxidant capacity, and sensitized to therapy. In this setting, fl-E6 repressed the PGC-1/ERR pathway for mitochondrial biogenesis by reducing p53-dependent PGC-1 transcription. Concordant observations were made in three clinical cohorts, where expression of mitochondrial components was higher in tumors of patients with reduced survival. These tumors contained the lowest fl-E6 levels, highest p53 target gene expression, and an activated PGC-1/ERR pathway. Our findings demonstrate that E6 can potentiate treatment responses by depleting mitochondrial antioxidant capacity and provide evidence for low E6 negatively impacting patient survival. E6s interaction with the PGC-1/ERR axis has implications for predicting and targeting treatment resistance in OPSCC. | cancer biology |
10.1101/2022.05.04.490656 | Lipid droplets are a metabolic vulnerability in melanoma | Melanoma exhibits numerous transcriptional cell states including neural crest-like cells as well as pigmented melanocytic cells. How these different cell states relate to distinct tumorigenic phenotypes remains unclear. Here, we use a zebrafish melanoma model to identify a transcriptional program linking the pigmented cell state to a dependence on lipid droplets, the specialized organelle responsible for lipid storage. Single-cell RNA-sequencing of these tumors show a concordance between genes regulating pigmentation and those involved in lipid and oxidative metabolism. This state is conserved in human melanoma specimens. This state demonstrates increased fatty acid uptake, an increased number of lipid droplets, and dependence upon oxidative metabolism. Genetic and pharmacologic suppression of lipid droplet production is sufficient to disrupt oxidative metabolism and slow melanoma growth in vivo. Because the pigmented cell state is linked to poor outcomes in patients, these data indicate a metabolic vulnerability in melanoma that depends on the lipid droplet organelle. | cancer biology |
10.1101/2022.05.04.490445 | Hypereosinophilia causes progressive cardiac pathologies in mice | Hypereosinophilic syndrome is a progressive disease with extensive eosinophilia that results in organ damage. Cardiac pathologies are the main reason for its high mortality rate. A better understanding of the mechanisms of eosinophil-mediated tissue damage would benefit therapeutic development. Here, we describe the cardiac pathologies that developed in a mouse model of hypereosinophilic syndrome. These IL-5 transgenic mice exhibited decreased left ventricular function at a young age which worsened with age. Mechanistically, we demonstrated infiltration of activated eosinophils into the heart tissue that led to an inflammatory environment. Gene expression signatures showed tissue damage as well as repair and remodeling processes. Cardiomyocytes from IL-5Tg mice exhibited significantly reduced contractility relative to WT controls. This impairment may result from the inflammatory stress experienced by the cardiomyocytes and suggest that dysregulation of contractility and Ca2+ reuptake in cardiomyocytes contributes to cardiac dysfunction at the whole organ level in hypereosinophilic mice.
TeaserToo many eosinophils cause inflammation in the heart and change cardiomyocyte contraction leading to poor heart function. | pathology |
10.1101/2022.05.04.490011 | Different combinations of laccase paralogs non-redundantly control the lignin amount and composition of specific cell types and cell wall layers in Arabidopsis | Vascular plants reinforce the cell walls of the different xylem cell types with lignin, a phenolic polymer. Specific lignin chemistries are conserved between the cell wall layers of each cell type to support their functions. Yet the mechanisms controlling the tight spatial localisation of specific lignin chemistries remain unclear. Current hypotheses focus on a control by monomer biosynthesis and/or export, while their cell wall polymerisation is viewed as random and non-limiting. Here we show that cell wall polymerisation using combinations of multiple different laccases (LACs) non-redundantly and specifically control the lignin chemistry in different cell types and their distinct cell wall layers. We dissected the roles of Arabidopsis thaliana LAC4, 5, 10, 12 and 17 by generating quadruple and quintuple loss-of-function mutants. Different combinatory loss of these LACs lead to specific changes in lignin chemistry affecting both residue ring structures and/or aliphatic tails in specific cell types and cell wall layers. We moreover showed that the LAC-mediated lignification had distinct functions in specific cell types. Altogether, we propose that the spatial control of lignin chemistry depends on different combinations of LACs with non-redundant activities immobilised in specific cell types and cell wall layers. | plant biology |
10.1101/2022.05.04.490655 | Introgression of the Triticum timopheevii genome into wheat detected by chromosome-specific KASP markers | Triticum timopheevii (2n=28, AtAtGG) is a tetraploid wild relative species with great potential to increase the genetic diversity of hexaploid wheat Triticum aestivum (2n=42, AABBDD) for various important agronomic traits. A breeding scheme that propagated advanced backcrossed populations of wheat-T. timopheevii introgression lines through further backcrossing and self-fertilisation resulted in the generation of 99 introgression lines (ILs) that carried 309 homozygous segments from the At and G subgenomes of T. timopheevii. These introgressions contained 89 and 74 unique segments from the At and G subgenomes, respectively. These overlapping segments covered 98.9% of the T. timopheevii genome that has now been introgressed into bread wheat cv. Paragon including the entirety of all T. timopheevii chromosomes via varying sized segments except for chromosomes 3At, 4G and 6G. Homozygous ILs contained between one and eight of these introgressions with an average of three per introgression line. These homozygous introgressions were detected through the development of a set of 480 chromosome-specific Kompetitive allele specific PCR (KASP) markers that are well-distributed across the wheat genome. Of these, 149 were developed in this study based on single nucleotide polymorphisms (SNPs) discovered through whole genome sequencing of T. timopheevii. A majority of these KASP markers were also found to be T. timopheevii subgenome specific with 182 detecting At subgenome and 275 detecting G subgenome segments. These markers showed that 98% of the At segments had recombined with the A genome of wheat and 74% of the G genome segments had recombined with the B genome of wheat with the rest recombining with the D genome of wheat. These results were validated through multi-colour in situ hybridisation analysis. Together these homozygous wheat-T. timopheevii ILs and chromosome-specific KASP markers provide an invaluable resource to wheat breeders for trait discovery to combat biotic and abiotic stress factors affecting wheat production due to climate change. | plant biology |
10.1101/2022.05.04.490425 | A marked enhancement of a BLOC-1 gene, pallidin, associated with somnolent mouse models deficient in histamine transmission | Histamine (HA) and orexin (Ox, or hypocretin) neurons act distinctly and synergistically in wake control. A double knock out mouse genotype lacking both HA and Ox shows all sleep disorders of human narcolepsy. We identified in this mouse brain a sharp upregulation of a BLOC-1 gene, pallidin that is associated with dramatic changes in the balance of cholinergic and aminergic systems in mice and an enhanced sleep in drosophila. This study demonstrates potential sleep disorders-associated compensatory mechanisms with pallid as a novel biomarker. | neuroscience |
10.1101/2022.05.04.490667 | The effects of physical and temporal certainty on locomotion with discrete underfoot perturbations | BackgroundAmbulation over complex terrain requires active control of foot placement to maintain a normal kinematic relationship between the center of mass and base of support. Recent investigations have suggested that foot placement location may be selected to anticipate shifts to the underfoot center of pressure. However, it is unclear whether temporal affordance and physical certainty contribute to the selection of a perturbation-specific anticipatory strategy. This study investigates anticipatory and reactive locomotor strategies for repeated underfoot perturbations with varying levels of temporal certainty, temporal affordance, and physical certainty.
MethodsThirteen healthy adults walked with random underfoot perturbations from a mechanized shoe. Temporal certainty was challenged by presenting the perturbations with or without warning. Temporal affordance was challenged by adjusting the timing of a warning tone before the perturbation. Physical certainty was challenged with conditions that included only eversion perturbations, only inversion perturbations, or both eversion and inversion perturbations. Linear-mixed effects models assessed the effect of each condition on the percent change of margin of stability and step width, respectively.
ResultsFor temporally uncertain perturbations and perturbations with one stride or less of affordance, we observed few changes to step width or margin of stability. As affordance increased to two strides, participants adopted a wider step width in anticipation of the perturbation (p = 0.001). Physical certainty had little effect on gait for the step of the perturbation, but participants recovered normal gait sooner when the physical nature of the perturbation was predictable (p < 0.001).
DiscussionDespite having information about the timing and magnitude of upcoming perturbations, individuals do not develop perturbation specific feedforward strategies but instead rely on feedback control to recover normal gait after a perturbation. However, physical certainty appears to improve the efficiency of the feedback controller and allows individuals to recover normal gait sooner. | neuroscience |
10.1101/2022.05.04.490623 | Katanin is involved in Microtubule Polymerization into Dendritic Spines and regulates Synaptic Plasticity | Dynamic microtubules transiently polymerize into dendritic spines, however intracellular factors that regulate this process and their functional role at synapses are hardly understood. Using live imaging, electrophysiology, and glutamate uncaging, we show that the microtubule-severing complex katanin is located at individual spine synapses, participates in the activity-dependent process of microtubule polymerization into dendritic spines, and regulates synaptic plasticity. Overexpression of a dominant-negative ATPase-deficient katanin subunit, did not alter microtubule growth velocities or comet density in dendrites, but significantly reduced the activity-dependent invasion of microtubules into dendritic spines. Notably, functional inhibition of katanin significantly affected the potentiation of AMPA-receptor-mediated excitatory currents after chemical induction of long-term potentiation (cLTP). Furthermore, interference with katanin function prevented structural spine remodeling following single spine glutamate uncaging. Our data identify katanin at individual spine synapses in association with PSD-95. Thus, katanin regulates postsynaptic microtubules and modulates synaptic structure and function. | neuroscience |
10.1101/2022.05.04.490609 | Population coding strategies in human tactile afferents | Sensory information is conveyed by populations of neurons, and coding strategies cannot always be deduced when considering individual neurons. Moreover, information coding depends on the number of neurons available and on the composition of the population when multiple classes with different response properties are available. Here, we study population coding in human tactile afferents by employing a recently developed simulator of mechanoreceptor firing activity. First, we demonstrate that the optimal afferent density for conveying maximal information depends on the tactile feature under consideration and the afferent class coding this feature. Second, we find that information is spread across different classes for all tactile features, such that combining information from multiple afferent classes improves information transmission, and is often more efficient than increasing the density of afferents from the same class. Finally, we test the importance of timing precision and afferent identity in the population code to probe whether temporal and spatial information can be traded against each other. Destroying temporal information turns out to be more destructive than removing spatial information, and the contribution of either cannot be completely recovered from the other. Overall, our results suggest that both optimal afferent innervation densities and the composition of the population depend in complex ways on the tactile features in question, potentially accounting for the variety in which tactile peripheral populations are assembled in different regions across the body. | neuroscience |
10.1101/2022.05.04.490675 | Actin nano-architecture of phagocytic podosomes | Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterized the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy (iPALM), a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy (SIM) and localization-based superresolution microscopy. Phagocytic podosomes were observed during frustrated phagocytosis, a model in which cells attempt to engulf micro-patterned IgG antibodies. For circular patterns, this resulted in regular arrays of podosomes with well-defined geometry. Using persistent homology, we developed a pipeline for semi-automatic identification and measurement of podosome features. These studies revealed an "hourglass" shape of the podosome actin core, a protruding "knob" at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, -actinin, cortactin, and microtubules relative to actin were characterized. | cell biology |
10.1101/2022.05.04.490571 | A multiplexed assay of human glucokinase reveals thousands of potential disease variants with both decreased and increased activity | Diabetes is a complex disease spanning from the heterogeneous etiology of type 1 and type 2 diabetes to monogenic diabetes. A common monogenic form of diabetes is glucokinase (GCK) maturity-onset diabetes of the young (GCK-MODY), which is caused by heterozygous inactivating variants in the gene encoding GCK. GCK is known as the pancreatic glucose sensor, as it regulates insulin secretion to maintain appropriate blood glucose levels. Accordingly, variants that alter GCK activity can cause hypo- and hyperglycemia, associated with hyperinsulinemic hypoglycemia (HH) and GCK-MODY, respectively, affecting up to 10 million people worldwide. Patients with GCK-MODY, in contrast to other people with diabetes, often do not require treatment but are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. To address this, we generated a comprehensive map of GCK variant activity in yeast. The activity map includes 97% of the possible missense and nonsense variants and correlates with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and evolutionary conservation analysis. Activity scores include both hyper- and hypoactive variants. We found that some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. As expected, hypoactive variants were concentrated at buried positions, near the active site, and at a partially surface-exposed region involved in GCK conformational dynamics. In conclusion, we provide a comprehensive assessment of GCK variant activity to facilitate variant interpretation and diagnosis, and we expand the mechanistic understanding of hyperactive variants to support development and refinement of drugs targeting GCK. | genetics |
10.1101/2022.05.04.490676 | Contributions and synaptic basis of diverse cortical neuron responses to task performance | Neuronal responses during behavior are diverse, ranging from highly reliable classical responses to irregular or seemingly-random non-classically responsive firing. While a continuum of response properties is frequently observed across neural systems, little is known about the synaptic origins and contributions of diverse response profiles to network function, perception, and behavior. Here we use a task-performing, spiking recurrent neural network model incorporating spike-timing-dependent plasticity that captures heterogeneous responses measured from auditory cortex of behaving rodents. Classically responsive and non-classically responsive model units contributed to task performance via output and recurrent connections, respectively. Excitatory and inhibitory plasticity independently shaped spiking responses and task performance. Local patterns of synaptic inputs predicted spiking response properties of network units as well as the responses of auditory cortical neurons from in vivo whole-cell recordings during behavior. Thus a diversity of neural response profiles emerges from synaptic plasticity rules with distinctly important functions for network performance. | neuroscience |
10.1101/2022.05.04.490677 | Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation | Basal cells are multipotent stem cells of a variety of organs, and in the lung are known as crucial components of the airway epithelium. However, it remains unclear how diverse basal cells are and whether distinct subpopulations respond differently to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the murine trachea. Among these we found two major subpopulations comprising the most stem-like progenitor compartment, but with distinct signatures and ability to self-renew and differentiate. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to initiate an aberrant program of differentiation in response to environmental perturbations in primary cultures and in injury mouse models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of cartilage-deficient mouse mutants. Lastly, we show that key features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide critical insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions. | developmental biology |
10.1101/2022.05.04.490677 | Airway Basal Cells Show Regionally Distinct Potential to Undergo Metaplastic Differentiation | Basal cells are multipotent stem cells of a variety of organs, and in the lung are known as crucial components of the airway epithelium. However, it remains unclear how diverse basal cells are and whether distinct subpopulations respond differently to airway challenges. Using single cell RNA-sequencing and functional approaches, we report a significant and previously underappreciated degree of heterogeneity in the basal cell pool, leading to identification of six subpopulations in the murine trachea. Among these we found two major subpopulations comprising the most stem-like progenitor compartment, but with distinct signatures and ability to self-renew and differentiate. Notably, these occupy distinct ventral and dorsal tracheal niches and differ in their ability to initiate an aberrant program of differentiation in response to environmental perturbations in primary cultures and in injury mouse models in vivo. We found that such heterogeneity is acquired prenatally, when the basal cell pool and local niches are being established, and depends on the integrity of these niches, as supported by the altered basal cell phenotype of cartilage-deficient mouse mutants. Lastly, we show that key features that distinguish these progenitor subpopulations in murine airways are conserved in humans. Together, the data provide critical insights into the origin and impact of basal cell heterogeneity on the establishment of regionally distinct responses of the airway epithelium during injury-repair and in disease conditions. | developmental biology |
10.1101/2022.05.04.490696 | A Pairwise Imputation Strategy for Retaining Predictive Features When Combining Multiple Datasets | In the training of predictive models using high-dimensional genomic data, multiple studies worth of data are often combined to increase sample size and improve generalizability. A drawback of this approach is that there may be different sets of features measured in each study due to variations in expression measurement platform or technology. It is often common practice to work only with the intersection of features measured in common across all studies, which results in the blind discarding of potentially useful feature information that is measured only in individual or subsets of all studies. We characterize the loss in predictive performance incurred by using only the intersection of feature information available across all studies when training predictors using gene expression data from microarray and sequencing datasets. We study the properties of linear and polynomial regression for imputing discarded features and demonstrate improvements in the external performance of predictors through simulation and in gene expression data collected on breast cancer patients. We propose and evaluate a pairwise imputation strategy that imputes cross-study missing features in each pair of studies and averages imputed features across pairs. Finally, we provide insights on which subsets of intersected and study-specific features should be used so that missing-feature imputation best promotes cross-study replicability. All code with directions to reproduce results in this paper is available at https://github.com/YujieWuu/Pairwise_imputation | bioinformatics |
10.1101/2022.05.04.490172 | Discovery and characterization of a first-in-field transcription factor BRN2 inhibitor for the treatment of neuroendocrine prostate cancer. | The increased incidence of treatment-emergent neuroendocrine prostate cancer (NEPC) is particularly alarming as this diagnosis is associated with poor prognosis. Despite initial responses to platinum-based chemotherapy, relapses are common and there is no effective second line therapy for NEPC. We previously identified that neuronal transcription factor BRN2 (POU3F2) is a potent driver of neuroendocrine differentiation and an attractive target for NEPC. Utilizing a combination of in silico modeling and X-ray crystallography followed by structure-based lead optimization, we have developed the first potent, specific and orally bioavailable BRN2 inhibitor (B18-94), which inhibits the interaction between BRN2 and DNA. This loss of BRN2 on the chromatin drastically reduces its transcriptional output resulting in downregulation of several known targets in NEPC such as SOX2, ASCL1 and PEG10. Additionally, B18-94 reduces specifically cell proliferation specifically in multiple NEPC models with no effect on adenocarcinoma and other BRN2 negative prostate cancer models. Importantly, the consistency in the transcriptomic changes driven by B18-94 and or CRISPR/Cas9 mediated BRN2 knockout confirmed the on-target specificity, with both methods of BRN2 inhibition downregulating pathways involved in cellular plasticity and proliferation. Finally, we have demonstrated that B18-94, the first-in-field POU-domain transcription factor inhibitor, significantly reduced tumor growth in several NEPC xenograft models with no observable toxicity, suggesting potential for therapeutic intervention of NEPC. | cancer biology |
10.1101/2022.05.04.490589 | bi-axial orientation could explain range expansion in a migratory songbird | The likelihood of a new migratory route evolving is a function of the associated fitness payoff, and the probability that the route arises in the first place. Cross-breeding studies suggest that young birds migrate in a direction intermediate between their parents, though this would seemingly not explain how highly divergent migratory trajectories arise in apparently sympatric populations. It has been suggested that diametrically opposed reverse migratory trajectories might be surprisingly common, and if such routes were heritable it follows that they could underlie the rapid evolution of divergent migratory trajectories. Here, we used Eurasian blackcap (Sylvia atricapilla; blackcap) ringing recoveries and geolocator trajectories to investigate whether a recently-evolved northwards autumn migratory route could be explained by the reversal of each individuals expected southwards migratory direction. We found that northwards migrants were recovered closer to the sites specified by a precise axis reversal than would be expected by chance, consistent with the rapid evolution of new migratory routes via bi-axial variation in orientation. We suggest that the surprisingly high probability of axis reversal might allow birds to expand their wintering ranges rapidly, and hence propose that understanding how direction is encoded is crucial when characterising the genetic basis of migratory direction and how this relates to route evolution. | zoology |
10.1101/2022.05.04.490590 | Group A streptococci induce high-affinity M protein-fibronectin interaction when specific human antibodies are bound | Group A streptococcus (GAS) is a highly adapted, humanspecific pathogen that is known to manipulate the immune system through various mechanisms. GAS M protein constitutes a primary target of the immune system due to its spatial configuration and dominance on the bacterial surface. Antibody responses targeting the M protein have been shown to favor the conserved C region. Such antibodies circumvent antigenic escape and efficiently bind to various M types. The ability of GAS to bind to fibronectin (Fn), a high molecular weight glycoprotein of the extracellular matrix, has long been known to be essential for the pathogens evolutionary success and fitness. However, some strains lack the ability to efficiently bind Fn. Instead, they have been found to inefficiently bind Fn via the M protein A-B domains. Here, we show that human antibodies can induce a high-affinity Fn-binding state in M proteins, likely by enhancing the weak A-B domain binding. The antibodies bind to a conserved region of M proteins, and the high-affinity binding only occurs on the individual M proteins with bound specific antibodies. By allowing the binding of antibodies to a certain region in M, and thereby enhancing Fn-binding, GAS exploits the human humoral immune response to efficiently bind Fn without needing to waste energy on the production of additional proteins - potentially giving such strains an evolutionary advantage. | immunology |
10.1101/2022.05.04.490594 | Population Genomics of Stone Age Eurasia | The transitions from foraging to farming and later to pastoralism in Stone Age Eurasia (c. 11-3 thousand years before present, BP) represent some of the most dramatic lifestyle changes in human evolution. We sequenced 317 genomes of primarily Mesolithic and Neolithic individuals from across Eurasia combined with radiocarbon dates, stable isotope data, and pollen records. Genome imputation and co-analysis with previously published shotgun sequencing data resulted in >1600 complete ancient genome sequences offering fine-grained resolution into the Stone Age populations. We observe that: 1) Hunter-gatherer groups were more genetically diverse than previously known, and deeply divergent between western and eastern Eurasia. 2) We identify hitherto genetically undescribed hunter-gatherers from the Middle Don region that contributed ancestry to the later Yamnaya steppe pastoralists; 3) The genetic impact of the Neolithic transition was highly distinct, east and west of a boundary zone extending from the Black Sea to the Baltic. Large-scale shifts in genetic ancestry occurred to the west of this "Great Divide", including an almost complete replacement of hunter-gatherers in Denmark, while no substantial ancestry shifts took place during the same period to the east. This difference is also reflected in genetic relatedness within the populations, decreasing substantially in the west but not in the east where it remained high until c. 4,000 BP; 4) The second major genetic transformation around 5,000 BP happened at a much faster pace with Steppe-related ancestry reaching most parts of Europe within 1,000-years. Local Neolithic farmers admixed with incoming pastoralists in eastern, western, and southern Europe whereas Scandinavia experienced another near-complete population replacement. Similar dramatic turnover-patterns are evident in western Siberia; 5) Extensive regional differences in the ancestry components involved in these early events remain visible to this day, even within countries. Neolithic farmer ancestry is highest in southern and eastern England while Steppe-related ancestry is highest in the Celtic populations of Scotland, Wales, and Cornwall (this research has been conducted using the UK Biobank resource); 6) Shifts in diet, lifestyle and environment introduced new selection pressures involving at least 21 genomic regions. Most such variants were not universally selected across populations but were only advantageous in particular ancestral backgrounds. Contrary to previous claims, we find that selection on the FADS regions, associated with fatty acid metabolism, began before the Neolithisation of Europe. Similarly, the lactase persistence allele started increasing in frequency before the expansion of Steppe-related groups into Europe and has continued to increase up to the present. Along the genetic cline separating Mesolithic hunter-gatherers from Neolithic farmers, we find significant correlations with trait associations related to skin disorders, diet and lifestyle and mental health status, suggesting marked phenotypic differences between these groups with very different lifestyles. This work provides new insights into major transformations in recent human evolution, elucidating the complex interplay between selection and admixture that shaped patterns of genetic variation in modern populations. | evolutionary biology |
10.1101/2022.05.04.490594 | Population Genomics of Stone Age Eurasia | The transitions from foraging to farming and later to pastoralism in Stone Age Eurasia (c. 11-3 thousand years before present, BP) represent some of the most dramatic lifestyle changes in human evolution. We sequenced 317 genomes of primarily Mesolithic and Neolithic individuals from across Eurasia combined with radiocarbon dates, stable isotope data, and pollen records. Genome imputation and co-analysis with previously published shotgun sequencing data resulted in >1600 complete ancient genome sequences offering fine-grained resolution into the Stone Age populations. We observe that: 1) Hunter-gatherer groups were more genetically diverse than previously known, and deeply divergent between western and eastern Eurasia. 2) We identify hitherto genetically undescribed hunter-gatherers from the Middle Don region that contributed ancestry to the later Yamnaya steppe pastoralists; 3) The genetic impact of the Neolithic transition was highly distinct, east and west of a boundary zone extending from the Black Sea to the Baltic. Large-scale shifts in genetic ancestry occurred to the west of this "Great Divide", including an almost complete replacement of hunter-gatherers in Denmark, while no substantial ancestry shifts took place during the same period to the east. This difference is also reflected in genetic relatedness within the populations, decreasing substantially in the west but not in the east where it remained high until c. 4,000 BP; 4) The second major genetic transformation around 5,000 BP happened at a much faster pace with Steppe-related ancestry reaching most parts of Europe within 1,000-years. Local Neolithic farmers admixed with incoming pastoralists in eastern, western, and southern Europe whereas Scandinavia experienced another near-complete population replacement. Similar dramatic turnover-patterns are evident in western Siberia; 5) Extensive regional differences in the ancestry components involved in these early events remain visible to this day, even within countries. Neolithic farmer ancestry is highest in southern and eastern England while Steppe-related ancestry is highest in the Celtic populations of Scotland, Wales, and Cornwall (this research has been conducted using the UK Biobank resource); 6) Shifts in diet, lifestyle and environment introduced new selection pressures involving at least 21 genomic regions. Most such variants were not universally selected across populations but were only advantageous in particular ancestral backgrounds. Contrary to previous claims, we find that selection on the FADS regions, associated with fatty acid metabolism, began before the Neolithisation of Europe. Similarly, the lactase persistence allele started increasing in frequency before the expansion of Steppe-related groups into Europe and has continued to increase up to the present. Along the genetic cline separating Mesolithic hunter-gatherers from Neolithic farmers, we find significant correlations with trait associations related to skin disorders, diet and lifestyle and mental health status, suggesting marked phenotypic differences between these groups with very different lifestyles. This work provides new insights into major transformations in recent human evolution, elucidating the complex interplay between selection and admixture that shaped patterns of genetic variation in modern populations. | evolutionary biology |
10.1101/2022.05.02.490346 | BREEDIT: A novel multiplex genome editing strategy to improve complex quantitative traits in maize (Zea mays L.) | Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Though new technologies are being applied to tackle the problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in gene engineering via the CRISPR/Cas technology pave the way to accelerate plant breeding and meet this increasing demand. Here, we present a gene discovery pipeline named BREEDIT that combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought resistance. We induced gene knockouts in 48 growth-related genes using CRISPR/Cas9 and generated a collection of over 1000 gene-edited maize plants. Edited populations displayed, on average, significant increases of 5 to 10% for leaf length and up to 20% for leaf width compared with controls. For each gene family, edits in subsets of genes could be associated with increased traits, allowing us to reduce the gene space needed to focus on for trait improvement. We propose BREEDIT as a gene discovery pipeline which can be rapidly applied to generate a diverse collection of mutants to identify subsets of promising candidates that could be later incorporated in breeding programs. | systems biology |
10.1101/2022.05.04.490569 | Development of a Well-Characterized Cynomolgus Macaque Model of Sudan Virus Disease for Support of Product Development | The primary objective of this study was to characterize the disease course in cynomolgus macaques exposed to Sudan virus (SUDV), to determine if infection in this species is an appropriate model for the evaluation of filovirus countermeasures under the FDA Animal Rule. Sudan virus causes Sudan virus disease (SVD), with an average case fatality rate of approximately 50%, and while research is ongoing, presently there are no approved SUDV vaccines or therapies. Well characterized animal models are crucial for further developing and evaluating countermeasures for SUDV. Twenty (20) cynomolgus macaques were exposed intramuscularly to either SUDV or sterile phosphate buffered saline; 10 SUDV-exposed animals were euthanized on schedule to characterize pathology at defined durations post-exposure and 8 SUDV-exposed animals were not part of the scheduled euthanasia cohort. Survival was assessed, along with clinical observations, body weights, body temperatures, hematology, clinical chemistry, coagulation, viral load (serum and tissues), macroscopic observations, and histopathology. There were statistically significant differences between SUDV-exposed animals and mock-exposed animals for 26 parameters, including telemetry body temperature, clinical chemistry parameters, hematology parameters, activated partial thromboplastin time, serum viremia, and biomarkers that characterize the disease course of SUDV in cynomolgus macaques. | microbiology |
10.1101/2022.05.03.490538 | Online Bayesian Analysis with BEAST 2 | AO_SCPLOWBSTRACTC_SCPLOWThere are a growing number of areas, e.g. epidemiology and within-organism cancer evolution, where re-analysing all available data from scratch every time new data becomes available or old data is refined is no longer feasible. All these and related areas can benefit from online phylogenetic inference that can booster previous data analyses.
Here, we make the case that adding/removing taxa from an analysis can have substantial non-local impact on the tree that is inferred, both in a model based setting, as well as for distance based methods. Consequently, online phylogenetic algorithms may require global updates of the trees and other parameters, a task that in general is highly non-trivial.
Motivated by this observation, we designed an online algorithm that benefits from a parallelism in a Bayesian setting that is substantially more efficient than re-running the analysis from scratch. Furthermore, our algorithm is not sensitive to the number of sequences added, allowing the sequence data to grow/be refined iteratively. We show how this approach can be used in a maximum likelihood setting, and - apart from adding/removing new sequences - demonstrate a number of practical alternative use cases of our algorithm, including how to break up a single (offline) large analysis to get results faster.
An open source implementation is available under GPL3 license as the online package for BEAST 2 at https://github.com/rbouckaert/online and a tutorial at https://github.com/rbouckaert/online-tutorial. | bioinformatics |
10.1101/2022.05.03.490539 | Human collision avoidance behavior against autonomous mobile robot | BackgroundAs urban development toward smart cities continues in earnest, pedestrians chances of encountering autonomous mobile robots (AMRs) on the street increase. Although recent studies have discussed how humans avoid collisions with others when passing them, it is still unclear how they would avoid AMRs, which could be common on the streets soon.
Research questionWe investigated humans avoidance strategy against an AMR approaching head-on through an experiment that included recording human-body motions while walking.
MethodThe AMR approached from various starting points, including directly from the participants. The participants were asked to circumvent it by moving rightward or leftward while their walking trajectories were tracked.
ResultWe found no significant bias on either side, suggesting that the avoidance direction is not simply determined by the participants attributes, such as the traffic rules followed in their area of living. The probability of rightward avoidance when the AMR approached head-on indicated that the humans had different avoidance strategy when facing other humans and objects. Moreover, the participants motion analysis revealed that their waists unconsciously twisted in the direction of avoidance before they circumvented.
SignificanceThe results suggest that the human-waist provides an indicator to predict the avoidance direction. Our findings could be adopted in AMRs development to fit them more naturally into our lives.
HighlightsO_LIHuman avoidance strategies against a robot approaching head-on were examined.
C_LIO_LIThe avoidance direction was not simply determined by the participants attributes.
C_LIO_LIHumans used different avoidance strategies when facing other humans and objects.
C_LIO_LIThe human waist provides an indicator to predict the avoidance direction.
C_LI | neuroscience |
10.1101/2022.05.04.490540 | Responses of Model Cortical Neurons to Temporal Interference Stimulation and Other Transcranial Alternating Current Stimulation Modalities | Temporal interference stimulation (TIS) has been proposed as a non-invasive, focal, and steerable deep brain stimulation method. TIS is hypothesized to activate neurons via the amplitude-modulated envelope of interference generated by two high-frequency (few kHz) sinusoidal electric fields (E-fields). Existing studies, however, oversimplified TIS and have not represent the full spatial dimensions of E-fields and cortical neurons. The response to TIS and other transcranial alternating current stimulation was simulated using detailed models of layer 5 pyramidal neurons adapted from the Blue Brain Project. We examined a wide range of parameter combinations, including the two E-fields orientations, frequencies, amplitude ratios, amplitude modulation, and phase difference, and obtained thresholds for both activation and inactivation, when stimulus-induced firing stops due to high stimulation amplitude.
TIS has a unique combination of characteristics. At the target region in the cortex, which is generally considered to be where the two E-fields have similar amplitudes, TIS generates an amplitude-modulated total E-field. The TIS E-field also exhibits rotation where the E-field orientations are not aligned, which generally co-localizes with the target. Outside the target region, the TIS E-field is dominated by the high-frequency carrier, with minimal amplitude modulation and/or rotation, and it is less effective at activation with low amplitudes and more effective at inactivation with high amplitudes. TIS activation thresholds are similar to high-frequency stimulation with or without modulation and/or rotation (75-230 V/m). TIS creates inactivation for some combinations of E-field orientations and amplitude ratios at high amplitudes (>1700 V/m), whereas amplitude modulated single-carrier high-frequency stimulation cannot achieve similar effects, regardless of orientation. All observed effects occurred at E-field strengths that are too high to be delivered tolerably through scalp electrodes, limiting the significance of suprathreshold TIS. | bioengineering |
10.1101/2022.05.04.490685 | Involvement of Lateral Habenula Dysfunction in Repetitive Mild Traumatic Brain Injury-Induced Motivational Deficits | Affective disorders including depression (characterized by reduced motivation, social withdrawal and anhedonia), anxiety and irritability are frequently reported as long-term consequences of mild traumatic brain injury (mTBI)1 in addition to cognitive deficits, suggesting a possible dysregulation within mood/motivational neural circuits. One of the important brain regions that control motivation and mood is the lateral habenula (LHb) whose hyperactivity is associated with depression2. Here we used a repetitive closed head injury mTBI model that is associated with social deficits in adult male mice3 and explored the possible long-term alterations in LHb activity and motivated behavior 10-14 days post-injury. We found that mTBI increased the proportion of spontaneous tonically active LHb neurons while decreased LHb bursting. Additionally, mTBI diminished spontaneous glutamatergic and GABAergic synaptic activity onto LHb neurons, while synaptic excitation and inhibition (E/I) balance was shifted toward excitation through a greater suppression of GABAergic transmission. Behaviorally, mTBI increased the latency in grooming behavior in sucrose splash test suggesting reduced self-care motivated behavior following mTBI. To show whether limiting LHb hyperactivity could restore motivational deficits in grooming behavior, we then tested the effects of Gi (hM4Di)-DREADD-mediated inhibition of LHb activity in sucrose splash test. We found that chemogenetic inhibition of LHb glutamatergic neurons was sufficient to reverse mTBI-induced delays in grooming behavior. Overall, our study provides the first evidence for persistent LHb neuronal dysfunction due to an altered synaptic integration as causal neural correlates of dysregulated motivational states by mTBI. | neuroscience |
10.1101/2022.05.04.490626 | Early structural connectivity within the sensorimotor network: deviations related to prematurity and association to neurodevelopmental outcome | The sensorimotor (SM) network is crucial for optimal neurodevelopment. However, undergoing rapid maturation during the perinatal period, it is particularly vulnerable to preterm birth.
Our work explores the prematurity impact on the microstructure and maturation of primary SM white matter (WM) tracts at term-equivalent age (TEA) and evaluates the relationships between these alterations and neurodevelopmental outcome.
We analyzed diffusion MRI data from the developing Human Connectome Project (dHCP) database: 59 preterm (PT) low-risk infants scanned near TEA, compared to a control group of full-term (FT) neonates paired for age at MRI and sex. We dissected pairwise connections between primary SM cortices and subcortical structures using probabilistic tractography and evaluated their microstructure with diffusion tensor imaging (DTI) and neurite orientation dispersion and density imaging (NODDI) models. In addition to tract- specific univariate analyses of diffusion metrics, we computed a maturational distance related to prematurity based on a multi-parametric Mahalanobis distance of each PT infant relative to the FT group. Finally, we evaluated the relationships between this distance and Bayley Scales of Infant and Toddler Development (BSID-III) scaled scores at 18 months corrected age.
Our results confirm important microstructural differences in SM tracts between PT and FT infants, with effects increasing with lower gestational age at birth. Additionally, comparisons of maturational distances highlight that prematurity has a differential effect on SM tracts which follows the established WM caudo-rostral developmental pattern. Our results suggest a particular vulnerability of projections involving the primary sensorimotor cortices (S1) and of the most rostral tracts, with cortico-cortical and S1-Lenticular tracts presenting the highest alterations at TEA. Finally, NODDI-derived maturational distances of specific tracts seem related to fine motor and cognitive scores.
This study expands the understanding of the impact of early WM alterations in the emerging SM network on long-term neurodevelopment. In the future, related approaches have potential to lead to the development of neuroimaging markers for neurodevelopmental disorders, with special interest for subtle neuromotor impairments frequently observed in preterm-born children. | neuroscience |