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RESEARCH ARTICLE Open Access
Feasibility and comparative analysis of
Dirofilaria immitis microfilaria freezing and
fixation for student instruction and
assessment of clinical parasitology skills
Sidney A. Long1, Jaylyn Rhinehart2, Jessica Shrake1and Antoinette E. Marsh1*
Abstract
Background: Detection of D. immitis microfilaria (mf) is an important diagnostic skill in veterinary medicine and is
critical to Day 1 veterinarians and technicians. Finding a supply of blood containing mf to teach the technique and
formalin ’s adverse environmental effects used in the diagnostic microscopic tests present a challenge.
Results: This study evaluated the use of cryopreserved and recently drawn mf-infected blood along with two
fixative reagents, acetic acid or formalin for mf detection. The specific aims included determining if veterinary
students could 1) detect cryopreserved mf added to fresh blood using routine diagnostic testing and 2) detect
morphological differences in the mf. The 236 students were kept blind from the sample status. The ability of the
students to identify mf and the mf morphology were compared for the samples and fixatives evaluated. The results
demonstrate using a combination of cryopreservation and acetic acid for teaching microfilaria diagnostic
techniques is fleasible; however, the quality of the mf morphology is less than optimal when compared to freshly
acquired mf containing blood. Compared to reference values, the mf demonstrated a decrease in size with each
additional variable evaluated.
Conclusion: A majority (98.3%) of the 236 students correctly identified the presence of mf. Teaching laboratories
could utilize cryopreserved mf-spiked donor blood in lieu of freshly collected mf-containing blood from a naturally
or experimentally infected dog. Substitution of less hazardous chemicals for the fixative can be used. Finally, the
change in size measurements provides a mechanism to ensure students can correctly measure mf as students are
required to do verifiable measurements and cannot copy reference values from a text book since the
cryopreservation and fixation methods cause the mf to measure smaller than textbook reference values.
Keywords: Heartworm, Dirofilaria immitis , Teaching, Mf, Cryopreservation, Diagnostics
Background
Dirofilaria immitis , the causative agent in heartworm
disease, is spread worldwide through infected mosquitos.
The main reservoir for D. immitis is the domestic dog,
but human, felid, and wildlife infections are documented
[1]. In 2018, the Companion Animal Parasite Council
compiled 143,492 cases of heartworm disease in dogs.
This is a significant increase in heartworm positive casesfrom just 5 years prior when in 2013, 77,557 cases of
heartworm disease in dogs were reported (CAPC Data-
base). Both climate change and increases in the inter-
state and international transport of dogs have increased
the prevalence of heartworm disease around the world
[1,2]. Microfilaremic dogs can serve as a reservoir for
parasite transmission. Finally, cases are costly to treat
and treatments can be associated with deleterious side
effects. All of these factors increase the need for veteri-
narians to be able to correctly diagnose D. immitis infec-
tion [ 1]. Diagnosis of canine dirofilariasis is critical to
public health as humans can be an incidental host of D.
© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated.* Correspondence: [email protected]
1Department of Veterinary Preventive Medicine, College of Veterinary
Medicine, The Ohio State University, 1920 Coffey Road, Columbus, OH 43210,
USA
Full list of author information is available at the end of the article
Long et al. BMC Veterinary Research (2020) 16:31
https://doi.org/10.1186/s12917-020-2248-3 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0000 |
immitis and suffer from clinical signs related to infection
[3].
Dirofilaria immitis diagnostics usually includes an
antigen test [ 1]. However false negatives due to
antibody-antigen blocking can occur [ 4]. The import-
ance of knowing how to test for microfilariae (mf) in
blood samples is critical for veterinarians and techni-
cians. Two main diagnostic techiques exist for mf detec-
tion: the Modified Knott ’s and carbonate filter tests [ 5].
The competency of veterinarians and technicians to per-
form these tests and recognize mf is reliant on the
teaching of the techniques during their clinical labora-
tory skills education. Often a supply of D. immitis mf in-
fected blood can be difficult to obtain when scheduled
to teach these diagnostic skills. To address the need for
a consistent source of D. immitis mf, cryopreservation of
the mf was evaluated. Successful cryopreservation of D.
immits mf would allow instructors to have a stock of mf
in the laboratory freezer for teaching purposes, without
having to obtain recently drawn “fresh ”mf containing
blood in sufficient volume when students want to prac-
tice diagnostic techniques.
Not only is banking the source for D. immitis mf im-
portant, but the use of “environmentally friendly ”fixatives
creates a need for evaluating modifications to diagnostic
teaching and the materials used in these assays. Both the
Modified Knott ’s and the carbonate filter procedures use a
2% formalin fixative [ 5]. Formalin is labeled as hazardous
waste in most laboratories due to the toxicity to humans
and the environmental implications [ 6]. Initial research on
comparing 2% formalin versus glacial acetic acid in a clin-
ical setting when performing the Modified Knott ’st e s t
suggests that 2% acetic acid can be substituded in place of
2% formalin fixation for easier disposal of the liquid waste
and lower potential health risks [ 7]. Therefore, this study
reports on the evaluation of mf cryopreservation tech-
niques used in conjunction with environmentally friendly
2% acetic acid in a teaching laboratory used for training
and assessment of veterinary students.
Results
Year 1 evaluating fleasibility of cryopreservation and
spiking donor blood
The mf that were recovered on days 0, 7, 21, and 70 day
remained above 897 mf per 50 ul of Modified Knott ’st e s t
(Table 1). The size of the mf varied and were smaller than
reference values 280-320 μm×6 . 1 –7.2μm (8) and 295-
325μm×5 . 0 –7.5μm[5]. Both isolates experienced a de-
crease in head scoring the longer the mf were frozen. There
was also a decrease in average mf concentration following
cryopreservation and then the concentration remained rela-
tively static thereafter (Fig. 1). Throughout the freezing time
evaluation, the JYD isolate had a greater concentration of
mf per ml as compared to the MO isolate.The average length and width of cryopreserved mf mea-
sured by 38 pairs of veterinary students was 260.5 μmb y
7.7μm. The average length and width of fresh mf mea-
sured by 21 pairs of veterinary students was 292.3 μmb y
12.2μm. All of the 118 veterinary students correctly iden-
tified mf in c-mf+. Two students commented that the mf
in c-mf + were smaller than expected and seven students
commented on the abundance of mf present.
During Year 1, a significant disadvantage occurred as
the Glycerolyte 57 solution inadvertently caused cryo-
preservation of the blood cells along with the mf in the
mf-infected whole blood. Later, these cryopreserved host
cells did not lyse well when exposed to the 2% formalin.
When the student performed the carbonate filter test,
the filter trapped intact non-lysed blood cells caused dif-
ficulties in expressing the solution through the filter as
compared to the nc-mf + samples. Many students also
commented about the excess amount of erythrocytes
present on the resulting slide-filter preparation.
Year 2 evaluating the preformanace of semi-purified frozen
microfilaria with either 2% acetic acid or 2% formalin
All of the 118 veterinary students correctly identified the
presence of mf in their c-mf + sample using 2% acetic acid
fixative. One pair of students reported a false negative
when evaluating a c-mf + sample using the 2% formalin
with the carbonate filter test. An instructor confirmed that
the carbonate filter had dried out under the microscope.
O n ep a i ro fs t u d e n t sr e p o r t e daf a l s en e g a t i v er e s u l tf r o ma
nc-mf + sample using the 2% acetic acid sample in the car-
bonate filter test. An instructor confirmed that the stu-
dents did not mix the blood sample and pipetted the blood
from the top of the sample. Microfilariae were found inTable 1 Modified Knott ’s test recovery comparison of D. immitis
microfilaria, MO and JYD isolates, using Year 1 cryopreservation
procedure
Day Isolate Recovery Measurements Size Score
0 MO > 1000 215.0 –255.0 × 2.5 –5 237.0 × 3 2.4
7 MO > 1000 257.5 –312.5 × 5 288.0 × 5 2.1
21 MO 1189 255.0 –300.0 × 5 276.8 × 5 2.2
70 MO 897 222.5 –270.0 × 2.5 –5 240.5 × 4.8 2.1
0 JYD > 1000 257.5 –292.5 × 5 274.0 × 5 3
7 JYD > 1000 257.5 –302.5 × 5 275.5 × 5 2
21 JYD > 1000 250.0 –292.5 × 5 271.8 × 5 2
70 JYD > 1000 237.5 –270.0 × 5 253.0 × 5 1.9
Day 0, no cryopreservation added, Day 7, 21, and 70 cryopreservation added
followed by freezing at −20 °C
MO and JYD isolates from experimentally infected dogs
Modified Knott ’s slide with 50-uL of sediment, number of microfilaria detected
Range for the 10 microfilaria measured in μm
Average length and width of the microfilaria measured in μm
Average Head Score- 1-3 scale, 3 = clear and distinguished head; 2 = stained
head but lighter than body; 1 = stained same color as body
or indistinguishableLong et al. BMC Veterinary Research (2020) 16:31 Page 2 of 8 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0001 |
the tube ’sr e s i d u a l~ 2 0 0u lo fb l o o ds a m p l eu s i n gt h e
Modified Knott ’s test following the release of the coded la-
boratory exercise. Two pairs of students that were given
mf- samples reported their samples as positive for mf using
the carbonate filter test. One pair measured their pur-
ported “mf”to be 690 μm and the other pair measured
their purported “mf”to be 84 μm.
The 2% acetic acid fixative caused the blood to turn sig-
nificantly darker than with 2% formalin. The 2% formalin
also created a darker staining of mf as compared to the
2% acetic acid fixation. The 2% acetic acid in combination
with c-mf + caused a greater decrease in the average
length of the mf compared to any other fixative/mf
combination.
During the initial phase to semi-purify and cryopreserve
the mf, the mixture of red blood cells and mf underwent a
lysis step prior to mf cryopreservation to mitigate the
problem of cryopreserved erythrocytes and blood cells ex-
perienced during Year 1. This modification improved theassay technique (less filter backpressure) and the quality
of the slides (fewer background erythrocytes). An un-
anticipated observation from the Year 2 technique of cryo-
preservation when stored at −80 °C included visualizing
motile mf in a drop of the thawed sample. However, the
time duration for mf to remain motile was not measured.
Table 2shows the average length and width reported by
the veterinary students and the experienced diagnosticians
for each assay type. The length and width followed the
same decreasing measurement value trend for both the di-
agnosticians and students in this order: nc-mf + in 2% for-
malin > nc-mf + in 2% acetic acid > c-mf + in 2% formalin
> c-mf + in 2% acetic acid.
Both experienced diagnosticians ranked a nc-mf + sam-
ple as the best overall morphology and slide quality. Over-
all, diagnostician A ranked the slides from best to worst in
the following order: nc-mf + in 2% formalin (best), then c-
mf + in 2% formalin, then nc-mf + in 2% acetic acid, and
c-mf + in 2% acetic acid (worst). Diagnostician B ranked
Fig. 1 Average number of microfilariae recovered per study timepoint, baseline, Day 0, Day 7, Day 21 and Day 70 after cryopreservation using
Year 1 protocol and the Modified Knott ’s test. Counts were preformed on 10 random microscope fields using the 10x objective
Table 2 Measurements in Year 2 using the Modified Knott ’s test
Assay type Diagnosticians ( n= 2) Students ( n= 118) n
length width length width
Non-cryopreserved mf, 2% formalin 306.2 5.5 302.8 8.3 22
Non-cryopreserved mf, 2% acetic acid 298.3 5.8 297.3 8.3 21
Cryopreserved mf, 2% formalin 281.2 5.5 277.8 6.4 23
Cryopreserved mf, 2% acetic acid 283.0 5.4 256.7 6.3 28
n- total number of students evaluating the sample and fixative
Measurements in micronsLong et al. BMC Veterinary Research (2020) 16:31 Page 3 of 8 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0002 |
the slides from best to worst in the following order: nc-
mf + in 2% acetic acid (best), then nc-mf + in 2% formalin,
then c-mf + in 2% formalin, and c-mf + in 2% acetic acid
(worst). Both diagnosticians made comments that the 2%
acetic acid stained lightly whereas the 2% formalin stained
more darkly, and that the nc-mf + samples created straigh-
ter mf as compared to the c-mf + samples.
Assessment of clinical students during their hospital rotation
All eleven of the fourth year clinical students identified
the c-mf + sample as mf+. Three of the eleven clinical stu-
dents identified the mf- sample as mf+. The measure-
ments of the purported “mf+ ”were 1500 μm×5 μm,
110μm×2 μm, and 450 μm×5 μm which indicates the
students were not actually looking at mf as that particular
sample contained none of the purported mf. There were 6
of the 11 students that attempted to identify the microfil-
aria with the following results: 3 identified Acanthocheilo-
nema reconditum , 2 identified D. immitis , and 1 identified
Dirofilaria repens . For true positive samples containing
the cryopreserved mf spiked sample, the average mf meas-
urement was 270.5 μm × 6.7 μm(n=1 1 ) .
Discussion
The MO and JYD isolates used contained a high concen-
tration of mf. Even with a low recovery of mf it is possible
for the students to obtain mf for identification if the start-
ing concentration of mf is high. Blood with a very low
concentration of mf may not be an adequate candidate for
cryopreservation. There is a drop in the number of mf re-
covered when the blood is cryopreserved compared to the
freshly acquired non-frozen mf-infected blood. Once cryo-
preserved, the concentration of mf recovered appears to
remain relatively stable over the time as evaluated in this
study (Fig. 1).
In the teaching laboratory veterinary students being
taught the diagnostic technique did not have difficulties
identifying the presence or absence of mf in the samples
regardless of cryopreservation, fixation, or technique in
either Years 1 or 2. The cryopreservation procedure con-
sistently caused a decrease in mf average length and
width compared to non-cryopreserved mf. This is a con-
cern for specifically teaching parasite identification based
on size because the reduced size of the mf led the stu-
dents to believe that the mf were not D. immitis but a
different parasite. Five pairs of students in the laboratory
course misidentified the mf and thought they were A.
reconditum based on size. Acanthocheilonema recondi-
tum length is 250 –288μm and width 4.5 –5.5μm[5].
Moreover, it must be considered that the students are
inexperienced with viewing the distinctive morphology
of the D. immitis mf, and this was the first time the stu-
dents performed these techniques in the teaching labora-
tory to view the mf microscopically. All five pairs ofstudents that identified the D. immitis mf as A. recondi-
tum had c-mf + and four of the pairs of students used
2% formalin while one pair of students used 2% acetic
acid. These students reported 220 –275μm for their
measurements. The students who reported false nega-
tives experienced technical difficulties as one student
pair allowed their filter (carbonate filter assay) to dry out
during their prolonged slide reading (> 45 min), and the
other student pair failed to properly mix the blood sam-
ple tube prior to preforming the assay. Thus, it is im-
portant for instructors to monitor the students
preforming the assays to ensure they are compliant with
standard operating procedures.
Cryopreserved mf do not straighten like non-cryopre-
served mf when exposed to fixative. This can lead to diffi-
culties in measuring the mf and may be responsible for
small discrepancies between student measurements. How-
ever, measurements of the mf using digital photomicro-
graphs with software to include measuring the mf curvature
produced similar measurements as the students (Fig. 2).
The mf recovered in Year 1 were given a head score and
cuticle score to compare cryopreserved mf that had been
frozen for different time points. The head and cuticle
scoring are both subjective data. The identification of mf
relies on measurements and the tail and head morphology
to distinguish species. The mf morphology quality score
and measured average length decreased following cryo-
preservation. However, the 124 day post-freezing samples
were sufficient for teaching the technique and allowing
visualization of the mf recovered despite issues with the
mf measurements and morphology.
A study evaluating the use of frozen-thawed human
tissues for suture technique education determined that
cryopreserved human tissues can serve as a teaching al-
ternative that provide similar characteristics to original,
natural tissues [ 8]. Practical techniques for mf cryo-
preservation were considered for use in a teaching la-
boratory similar to how human cryopreservation studies
have addressed the practical use of tissue [ 8,9]. The mf
were stored at −20 °C and at −80 °C with no detectable
changes in the mf. Most laboratories will have a −20 °C
freezer. Not all teaching laboratories will have access to
a−80 °C freezer or liquid nitrogen, and cryopreservation
needs to be applicable to general teaching facilities.
Easy disposal of waste after performing the assays led to
the inclusion of 2% acetic acid fixative in place of 2% for-
malin for easier disposal, safer handling, and as an envir-
onmentally friendly option. Water was initially evaluated
during the pilot freezing trial, but because of the variation
in measurements relative to reference values it was not
pursued for the student evaluation trials. Nonetheless, the
2% acetic acid did cause noticeable changes to the blood
coloration, mf-staining, and size as compared to the 2%
formalin. These issues should be addressed with theLong et al. BMC Veterinary Research (2020) 16:31 Page 4 of 8 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0003 |
students if the students, when in practice, plan to use 2%
formalin solution as specified in reference manuals for the
technique protocols. Therefore, further optimization of
the acetic acid percentage used with the c-mf + could re-
sult in better staining, but it may not completely resolve
the disparent size relative to associated reference values
when using 2% formalin with freshly drawn blood con-
taining mf.
Clinical students in their fourth year rotation could con-
fidently detect mf from a positive sample. A few students
detected artifact and identified the structures as mf despite
having a negative sample. All of the artifacts detected as
“microfilaria ”had measurements well out of the range for
normal mf, supporting that students are in need of more
practice to become more comfortable reading slides.
Many veterinary schools have incorporated clinical skills
laboratories into their program and cryopreservation to
bank a source of mf could allow students more opportun-
ities to practice these techniques. Ordering fresh mf in-
fected blood throughout the year for a clinical skills
laboratory is impractical, costly, and D. immitis infected
research dogs are limited in the frequency they can be
bled. Cryopreservation allows for less frequent bleeding
and a more consistent source of mf for teaching.
Conclusions
The majority of the 236 laboratory course students from
Year 1 and Year 2 were able to correctly detect mf froma“positive sample ”either their prepared slide or viewing
another pair ’s slide. All 28 students with the 2% acetic
acid and c-mf + combination correctly identified the
presence of mf. Of the 236 laboratory course students
that participated 234 (99.2%) were able to identify mf
from a c-mf + sample regardless of fixative or technique.
In some cases, they relied on another student pair ’s pre-
pared slide. Banking the source for mf and the practical
use of 2% acetic acid could improve the teaching of diag-
nostics for heartworm disease. Eleven students were
evaluated during their clinical hospital rotation. All the
students could perform the Modified Knott ’s assay but 3
out of 11 still reported false positive results. This
method is suitable as a stand-alone for practicing the
diagnostic technique but the teaching laboratory should
include a displayed slide of D. immitis mf representing
the correct size and good staining to display appropriate
morphology for students to compare for learning and
practicing parasite identification. In a clinical skills la-
boratory, the use of c-mf + with 2% acetic acid fixative
provides a reasonable alternative for teaching purposes
and evaluating students ’diagnostic skills.
Methods
Year 1 evaluating fleasibility of cryopreservation and
spiking donor blood
In Year 1, only the cryopreservation of D. immitis was
evaluated. Two isolates of infected blood were obtained
Fig. 2 Modified Knott ’s test slide preparations using variable testing conditions in Year 2 with microfilariae (mf) length and width measurements
in microns and variable background erythrocytes and host cells. a. Non-cryopreserved mf with 2% formalin fixative. b. Cryopreserved mf with 2%
formalin fixative. c. Non-cryopreserved mf with 2% acetic acid. d. cryopreserved mf with 2% acetic acidLong et al. BMC Veterinary Research (2020) 16:31 Page 5 of 8 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0004 |
from the NIH/NIAID Filariasis Research Reagent Re-
source Center, JYD and MO strains, collected into
heparized tubes and shipped overnight. One aliquot of
fresh blood was removed and designated as a baseline
and immediately evaluated using the Modified Knott ’s
test, [ 10] modified to use water to substitute for the 2%
formalin. Photomicrographs were taken and the Modi-
fied Knott ’s’preparation was permanently mounted onto
a slide. The remaining heparinized whole blood was
mixed 1:1 with Glycerolyte 57 solution (Baxter, USA),
divided into 1 ml aliquots and frozen at −20 °C. One
sample was then immediately thawed (Day 0) while the
others remained frozen for later diagnostic test process-
ing on days 7, 21, 70, and 124 post-freezing.
On days 7, 21, and 70 two samples of each isolate were
removed from the freezer to perform a Modified Knott ’s,
again using water instead of the 2% formalin. Total vol-
ume of pellets were measured for recovery purposes. New
methylene blue (Ricca Chemical Company, USA) was
used to stain the microfilaria for all of the Modified
Knott ’s and carbonate filter tests performed in this study.
The detected mf quantity and morphology were recorded
for each time point and compared to baseline. Quantity
data was collected by evaluating the total number of mf
per slide and the average number of mf per 10x objective
field. The average number of mf per field was found by
taking 10 random views of the slide and averaging the
number of mf seen. Morphology of ten individual mf was
evaluated using a head scoring of 1 –3, cuticle scoring of
1–3, and the measurements of the mf compared to refer-
ence values [ 5,8]. The head scoring parameters were 3-
clear and distinguished; 2-stained, but stained lighter than
the body; 1-indistinguished, head and body same color-
ation. The cuticle scoring parameters were 3-crisp and
defined edges of the body; 2-darkly stained; 1-unclear
edge/no edge or damaged. Photomicrographs were taken
at the different time points for comparison.
The day 124 post-freezing samples were used in the
teaching laboratory to run a blind trial on 118 veterinary
students. Instructors were also blinded. Pairs of students
received a randomly assigned unknown microfilaria-status
patient sample, coded by the investigator (SL), in a 3 ml
ethylenediaminetetraacetic acid disodium salt dehydrate
(EDTA) tube. The unknown patient samples consisted of
one of the following: non-cryopreserved mf infected blood
(nc-mf+), freshly acquired dog donor blood cells spiked
with cryopreserved-infected blood (c-mf+), or non-
infected normal dog donor blood cells (mf-). Acquisition
of the normal dog donor blood cells came from a donor
dog in the Ohio State Veterinary Medical Center Blood
Bank program. For the dog donor blood, the plasma was
removed and remaining blood cells were preserved using
Adsol red cell preservative solution (Fenwal, Inc., USA) in
the blood bank bag and kept at 4 °C.The samples and required testing assays were coded to
approximately distribute a variety of the samples and as-
says in each of the six laboratory sessions. The student
pairs randomly drew their assigned patient and method,
either a Modified Knott ’s or carbonate filter assay with
the standard 2% formalin fixation component. The stu-
dent pairs reported if their sample contained detectable
mf and if so, measured one or more mf on their slide. If
their sample contained no mf they were required to view
a positive slide from a different student pair and make
written observations about the slides. Students submit-
ted their written results worksheet for this exercise.
Year 2 evaluating the preformanace of semi-purified frozen
microfilaria with either 2% acetic acid to 2% formalin
In Year 2, further refinement of the study parameters
and a new set of 118 students were evaluated. Changes
included purification of mf prior to the addition of cryo-
protective medium and the use of 2% acetic acid as a
fixative in the techniques. Again the JYD and MO strains
ofD. immitis were used. For mf purification, heparinized
whole blood containing mf was centrifuged for 10 min at
400 G at 25 °C. The separated layers of plasma, buffy
coat, and red blood cells (RBC) were removed and a 10
ul aliquot was examined to check for mf. As the RBC
layer contained sufficient living mf, further processing of
this layer occurred. The 1 part of the RBC-mf layer was
mixed with 9 parts of water and gently inverted for 1
min to lyse the host cells. Physiological isomolarity was
returned using 9% sodium chloride solution. The mix-
ture underwent centrifugation for 10 min at 400 G to
pellet the mf, the resulting pellet was resuspended in an
equal volume of Glyerolyte 57, 200 ul aliquoted into in-
dividual cryovials, rapidly frozen on dry ice, then stored
at−20 °C until the day before class.
One day prior to the laboratory teaching session, the
mf samples were rapidly thawed at 38 °C and added to
pre-labeled 3 ml EDTA tubes containing 1 to 2 mls of
healthy donor dog blood cells. Similar to Year 1, the 118
blinded veterinary students were randomly assigned a
diagnostic technique (Modified Knott ’s or carbonate fil-
ter), a sample (nc-mf+, c-mf+, or mf-), and fixative (2%
acetic acid or 2% formalin) to analyze. Instructors and
students were kept blinded from the sample identity and
fixative. Fixative bottles were labeled A or B. Students
were paired with another student, and each pair ran-
domly drew a patient sample associated with an assigned
technique and fixative. Students were required to report
if mf were present, if so, measure at least one mf in their
slide, observe another student pair ’s prepared slide, and
make comments about the slides. We did not specifically
require students to identify the microfilaria to genus and
species. Based on the drawn sample ’s patient name, stu-
dents ’with c-mf + samples using the 2% acetic acidLong et al. BMC Veterinary Research (2020) 16:31 Page 6 of 8 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0005 |
fixative were sent to look at a c-mf + sample processed
with 2% formalin fixative and vice versa, so all students
compared the two different fixatives relative to their
microfilaria sample.
In Year 2 the samples were not evaluated on days 0, 7,
21, and 70 because the feasibility of storing cryopre-
served samples was determined in Year 1.
A blind trial was conducted with two experienced la-
boratory diagnosticians A and B. The Modified Knott ’s
procedure was used to prepare four slides which consisted
of the following: c-mf + in 2% acetic acid fixative, nc-mf +
in 2% acetic acid fixative, c-mf + in 2% formalin fixative,
and nc-mf + in 2% formalin fixative. The experienced di-
agnosticians were asked to rank the quality of the four
slides based on the morphology of and the ability to detect
mf. The best quality was ranked as 1 and the poorest over-
all as 4. The diagnosticians also measured two mf per
slide. Photomicrographs were taken using a Olympus
BX41 equipped with a DP74 camera (Olympus America
Inc., USA) connected to a Dell Optiplex 7050 (Dell Tech-
nologies, USA) with cellSens Standard 1.18 software pack-
age (Olympus America Inc) for image acquisition and
measurement tools.
Assessment of clinical students during their hospital
rotation
A blind trial was conducted on fourth year veterinary
students ( n= 11) during their clinical rotations. Two dif-
ferent student rotation groups were evaluated. This re-
quired the thawing and adding of cryopreserved
microfilaria to healthy donor dog blood cells in EDTA
tubes either 1 day or 14 days prior to the student assess-
ments. During this 2 week period the spiked blood was
kept at 4 °C. Each student was provided with 2% acetic
acid fixative and two “unknown labelled blood samples ”
with one being c-mf + (spiked) and the other mf- (nor-
mal dog donor blood cells). The students were
instructed to independently perform a Modified Knott ’s
on the blood samples. Students were required to report
on a submission form their results if mf were present,
and if so, measure and attempt to identify at least one
mf on their slide.
Ethics approval and consent The Ohio State University
Office of Responsible Research Practices Institutional
Review Board reviewed and exempted protocol
2019E0658 under 45 CFR 46 (USA). The research was
conducted in a commonly accepted educational setting,
involving normal educational practices and did not ad-
versely impact students ’opportunity to learn the re-
quired educational content. The research compared
reagents used for an instructional technique and was
therefore exempted from human subject research under
45 CFR 46.Abbreviations
mf:Microfilaria; nc-mf + : non-cryopreserved mf infected blood.; c-
mf + : freshly acquired donor blood cells spiked with cryopreserved-infected
blood.; mf-: non-infected normal dog donor red blood cells (mf-).;
mf + : microfilaria positive.; EDTA: ethylenediaminetetraacetic acid disodium
salt dehydrate
Acknowledgements
The authors acknowledge Drs. John Bonagura and Nina Zitzer for thoughtful
discussion, Cathy Bremer for technical assistance, and Marc Hardman for
illustration assistance. The following reagents were provided by the NIH/
NIAID Filariasis Research Reagent Resource Center for distribution by BEI
Resources, NIAID, NIH: Dirofilaria immitis , Strain MO, NR-48908, and Strain
JYD-27, NR-49172, live mf in dog blood.
Authors ’contributions
SL collected, analyzed, interpreted, and presented data from Year 1, Year 2,
and clinal students. SL was a major contributor to the manuscript. JR
organized the clinical students, conducted the blind trials for clinical
students and edited the manuscript. JS assisted with Year 1 data collection
and analysis. AM designed study, reviewed data and edited the manuscript.
All authors read and approved the final manuscript.
Funding
The Ohio State University College of Veterinary Medicine provided partial
financial support (Marsh for the shipping costs to receive the Dirofilaria
immitis parasites) and for the supplies used in the laboratory. The Ohio State
University Undergraduate Research Apprentice Program provided summer
stipend support (Long).
Availability of data and materials
Companion Animal Parasite Council Parasite Prevelance Maps for Heartworm
are publicly available at https://capcvet.org/maps/#2018/all/heartworm-
canine/dog/united-states/ . The datasets used and analyzed during the
current study are available from the corresponding author on reasonable
request.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1Department of Veterinary Preventive Medicine, College of Veterinary
Medicine, The Ohio State University, 1920 Coffey Road, Columbus, OH 43210,
USA.2Department of Veterinary Clinical Sciences, College of Veterinary
Medicine, The Ohio State University, 601 Vernon Tharp Drive, Columbus, OH
43210, 531, USA.
Received: 6 August 2019 Accepted: 20 January 2020
References
1. Bowman DD, Liu Y, McMahan CS, Nordone SK, Yabsley MJ, Lund RB.
Forecasting United States heartworm Dirofilaria immitis prevalence in dogs.
Parasit Vectors. 2016;9:540.
2. Wang D, Bowman DD, Brown HE, Harrington LC, Kaufman PE, McKay T,
Newlson CT, Sharp JL. Factors influencing U.S. canine heartworm ( Dirofilaria
immitis ) prevalence. Parasit Vectors. 2014;7:264.
3. Solgi R, Sadjjadi SM, Mohebali M, Zarei Z, Golkar M, Raz A. Development of
new recombinant DgK antigen for diagnosis of Dirofilaria immitis infections
in dogs using ELISA technique and its comparison to molecular methods.
Iran Biomed J. 2018;22(4):283 –9.
4. Little S, Raymond M, Thomas J, Gruntmeir J, Hostetler J, Meinkoth J,
Blagburn BL. Heat treatment prior to testing allows detection of antigen of
Dirofilaria immitis in feline serum. Parasit Vectors. 2014;7:1.
5. Zajac A, Conboy G. Veterinary clinical parasitology. 8th ed. Ames, Iowa:
Wiley Blackwell; 2012.Long et al. BMC Veterinary Research (2020) 16:31 Page 7 of 8 | ecf468dac39b30d6866baca4dd746f376c06b06c | page_0006 |
6. United States Department of Health and Human Services. National
Toxicology Program, 12thReport on carcinogens. Rep. Carcinog. 2011;12, iii –
499.
7. Evans CC, Bradner JL, Savadelis MD, Nelson CT, Moorhead AR. Acetic acid as
an alternative reagent in the modified Knott test. Vet Parasitol. 2019;276:
108975.
8. Song SY, Kang MK, Kim EK. Frozen-thawed abdominal flap remnant as an
education material for a medium group surgical skills education workshop.
Ann Surg Treat Res. 2019;96(2):53 –7.
9. Hohmann E, Keough N, Glatt V, Tetsworth K, Putz R, Imhoff A. The
mechanical properties of fresh versus fresh/frozen and preserved (Thiel and
formalin) long head of biceps tendons: a cadaveric investigation. Ann Anat.
2019;221:186 –91.
10. Foreyt W. Veterinary parasitology reference manual. 5th ed. Ames, Iowa:
Iowa State Univ. Press; 2001.
Publisher ’sN o t e
Springer Nature remains neutral with regard to jurisdictional claims in
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DOI:10.1002/hon.3006
LETTERTOTHEEDITOR
EffectivenessofathirddoseofBNT162b2anti‐SARS‐CoV‐2
mRNAvaccineovera6‐monthfollow‐upperiodinallogenic
hematopoietic stemcellsrecipients
Abstract
Thisstudyreportstheeffectiveness ofthreeinjections
ofBNT162b2 anti‐SARS‐CoV‐2 mRNAvaccine in141
Allo‐HSCT recipients withamedian follow‐up of
6monthspost‐third shot.Wedemonstrate along‐term
highprotection ofAllo‐HSCT recipients sinceonly2
infections andonedeathrelated toCOVID‐19
occurred.
Boosting immunity byproviding athirddoseofCOVID‐19 vaccine
hasnowbecome crucialinthegeneralpopulation asitnotonly
overcomes thewaningofhumoral immune responses afterafew
monthsbutalsorestores efficacyagainstnewvariants suchasDelta
andOmicron. Datareporting theinterest ofathird‐dose/boost
vaccine inimmunocompromized hosts,including solidtumorpa-
tients1orallogeneic hematopoietic stem‐cell transplant (allo‐HSCT)
recipients,2arealsoprogressively reported, yetwithveryshort
follow‐up.
SinceFrenchauthorities recommended boostsforhigh‐risk
patients inApril2021,wecanreportheretheeffectiveness of
thisstrategy in141allo‐HSCT recipients withamedianfollow‐up
of6months post‐third shot.Allreceived threeinjections of
BNT162b2 anti‐SARS‐CoV‐2 mRNAvaccine(V1V2V3)between
January andSeptember 2021.Themedianageofthecohortwas
58yearsold(range:20–77)andthemediandelaybetween the
graftandV1was33.5months(3–282), with28,33and80cases
withinthefirstyearpost‐transplant, thesecondyearoftransplant
orabove,respectively. Thestudywasapproved bythereview
boardofNantesUniversity Hospital andallparticipants provided
informed consent. Patientcharacteristics aregivenintheTable1.
Detectable antibody responses, testedtwiceafterV3(earlyS1
n=124,median33daysfromV3,andlateS2n=141,median
193daysfromV3)wereclassified as“weak”or“good”.AtS1and
S2,83%and82%ofthepatients hadgoodresponses, thatis,
abovethe250BAU/ml threshold, reported tocorrelate with
neutralizing antibody levels.2Forthe96patients testedtwicewith
thesameassay,theproportions ofgoodresponders remainedsimilarbetween S1(81%)andS2(79%),yetwithaslightdecrease
ofIgGtitersfor29%ofthem(Table1).Factors associated with
goodresponses were,asexpected,2ahigherlymphocyte (median
2.84�109/Lvs.1.00�109/L,p=0.005),CD4+Tcells(median
3.79�109/Lvs.2.05�109/L,p=0.001)andBcells(median
3.12�109/Lvs.0.68�109/L,p<0.001)countsatS2,absence of
immunosuppressive drugsorchemotherapy (82%vs.56%,
p=0.01),andbeingatleastoneyearpost‐transplant (85.3%vs.
56%,p=0.002).Effectiveness atS2orafterwasinvestigated in
termsofinfection, hospitalization andCOVID‐19‐related death.At
lastfollow‐up (18January 2022),andinlinewithpublished pre-
liminary results,2 only2mildCOVID‐19 infections occurred, both
inpatients withS2≥250BAU/ml (+168and +1042dayspost‐
transplant). TherewasoneCOVID‐19‐related death(S2:15.3
BAU/ml, +150dayspost‐transplant). Thedeathrateisthus0.7%
versus21%–25% fornon‐vaccinated allo‐HSCT recipients.3
Thisobservational studydemonstrates along‐term highpro-
tectionofAllo‐HSCT recipients vaccinated threetimeswiththe
BNT162b2 anti‐SARS‐CoV‐2 mRNAvaccine. Themediandelayof
44daysafterthesecondshotcouldbereconsidered asalongerin-
tervalbetween thesecondprimingdoseofvaccineandthebooster
doseappears toresultinhigherneutralizing antibody titersagainst
allvariants testedinarecentstudy.4
Waning ofIgGtitersconcerns around30%ofourpatients at
6monthsbutonlyasmallproportion (19%)hadIgGtiters <250
BAU/ml, suggesting thepossibility topropose afourthinjection to
enhance protection inthesecases.Ofnote,inhealthypopulation, a
6‐month long‐term follow‐up aftertheboosterhavebeenreported
showing thatneutralization titersagainsttheomicron variantwere
6.3timeslowerthanthepeaktitersassessed 1monthafterthe
booster injection, butthetitersremained detectable inallthe
participants.5
T‐cellimmunity maybealsointeresting toinvestigate atdis-
tanceoftheboostbutthisiscurrently challenging andnotper-
formedinroutinepractice. Finally,although theDeltavariantwas
predominant duringtheperiodofouranalyses, thelessergravity
ofOmicron suggests thatthesameresultsshouldbeobserved,
although thishypothesis hastobeconfirmed inthenextfew
months.
Hematological Oncology. 2022;1 –3. wileyonlinelibrary.com/journal/hon ©2022JohnWiley&SonsLtd.
-1 | a1493134f77670cb875d31694d924d9a52488db1 | page_0000 |
ACKNOWLEDGMENTS
Weacknowledge thefollowing individuals fortheirassistance with
thestudy,noneofwhomwascompensated forhisorhercontribu-
tions:Themedicalstaff:Drs.Berthe‐Marie Imbert,Thomas Drumel,
Beatrice Mahé,Viviane Dubruille, NicolasBlin,AnneLok,Cyrille
Touzeau, ThomasGastinne, SophieVanthygem, Philippe Moreauand
StevenLeGouillwhoprovided dataandcommented onthemanu-
script.Hematology Department nurses:Patricia Lespart, GhislaineTABLE1Patientcharacteristics, effectiveness andantibody
levels
PatientsN=141a
Medianage:years(range) 58(20–77)
<40/40–59/≥60 19/55/67
Gender: Male/Female 84/57
Underlyingdisease: Myeloid/Lymphoid 101/40
Donortype:sibling/MUD/haplo/9–10/cord blood 34/59/42/4/2
Conditioning: MAC/RIC/sequential 29/105/7
PreviousGVHD:Yes/no 77/64
Ongoingtreatment: Yes/no 32/109
Yes:Immunosuppressive drugs/Chemotherapy 27/5
MedianlymphocytecountatS2:Range(x109/L)1720(121–
6570)
</≥1�109/Ln= 32/109
Vaccineandserologydates
V1 Jan14th–15
June2021
V2 Feb4th–15July
2021
V3 Apr2nd–24Sept
2021
S1 May3rd–25Oct
2021
S2 Aug8th–18Jan
2022
Mediandelays
Graft‐V1: months(range) 33.5(3–282)
<6monthsn= 10
<12monthsn= 18
12–24monthsn= 33
>24monthsn= 80
V1‐V2:days(range) 23(12–52)
V2‐V3:days(range) 44(20–205)
V1‐S1:days(range) 121(76–242)
V1‐S2:days(range) 272(154–363)
Earlyserology(S1)afterV3N=124
Mediandelay:days(range) 33(13–139)
Tests
RocheStAb 92
AbbottSIgG 5
DiaSorin TriS 11
Atellica 13
Novalisa 3TABLE1(Continued)
PatientsN=141a
IgGtiters
Negative 14(11%)
Detectable <250BAU/ml 7(6%)
≥250BAU/ml 103(83%)
Lateserology(S2)afterV3N=141
Mediandelay:days(range) 193(94–263)
Techniques
RocheStAb 116
AbbottSIgG 7
DiaSorin TriS 14
Atellica 8
Novalisa 2
IgGtiters
Negative 13(9.5%)
Detectable <250BAU/ml 12(8.5%)
≥250BAU/ml 116(82%)
ComparisonofIgGtitersbetweenS1andS2
n=96b
Negative/negative 10(11%)
Decrease (<250BAU/ml/≥250 BAU/ml atS2) 28(9/19)(29%)
Increase (conversion) 4(1)(4%)
≥250BAU/ml atS1S2 54(56%)
Total ≥250BAU/ml atS1 78(81%)
Total ≥250BAU/ml atS2 76(79%)
Effectivenessofthethirdvaccinewithamedianof
6monthsfollow‐up
COVID‐19 infection 2(1.4%)
Hospitalization duetoCOVID‐19 infection 0
DeathfromCOVID‐19 1(0.7%)
Abbreviations: GVHD,graft‐versus‐host disease; haplo,haploidentical;
MAC,myeloablative; MUD,matched unrelated donor;RIC,reduced‐
intensity conditioning.
aIncluding 4withaprevious asymptomatic SARS‐CoV‐2 infection.
bUsingthesameserologic assaysatS1andS2forcomparison.2
/
LETTER TOTHEEDITOR | a1493134f77670cb875d31694d924d9a52488db1 | page_0001 |
Francois, andKatiaGodartforadministrating vaccines andtheirhelp
incollecting samples. Theparamedical staffoftheHematology
Department andoftheVirology Department.
CONFLICTOFINTEREST
Theauthorsdeclarenoconflictofinterest.
ETHICALSTATEMENT
Allprocedures followed wereinaccordance withtheethicalstan-
dardsoftheresponsible committee onhumanexperimentation
(institutional andnational) andwiththeHelsinki Declaration of1975,
asrevisedin2008.
AUTHORCONTRIBUTIONS
PatriceChevallier, Maxime JullienandThierryGuillaume designed,
performed, coordinated theresearch, analyzed, performed statistical
analyses, interpreted thedata,generated thefigure,andwrotethe
manuscript. Marianne Coste‐Burel performed serology tests,gener-
atedthevirologic dataandcommented onthemanuscript. Maxime
JullienandMarieC.Bénéperformed statistical analyses andcom-
mentedonthemanuscript.
STATEMENTOFINFORMEDCONSENT
Informed consent wasobtained fromallparticipants forbeing
included inthestudy.
PatriceChevallier1,2
Maxime Jullien1
PierrePeterlin1
AliceGarnier1
Amandine LeBourgeois1
Marianne Coste‐Burel3
MarieC.Béné2,4
ThierryGuillaume1,2
1Hematology Department, NantesUniversityHospital,Nantes,
France
2INSERMUMR1232, CRCINAIRS‐UN,UniversityofNantes,
Nantes,France3Virology0020Department, NantesUniversityHospital,Nantes,
France
4Hematology Biology,NantesUniversityHospital,Nantes,France
Correspondence
PatriceChevallier, Serviced’Hématologie Clinique, CHUHotel‐Dieu,
PlaceA.Ricordeau, 44093NantesCedex,France.
Email:[email protected]
DATAAVAILABILITY STATEMENT
Theprincipal investigator PChadfullaccesstoallthedatainthe
studyandtakesresponsibility fortheintegrity ofthedataandthe
accuracy ofthedataanalysis.
ORCID
PatriceChevallier
https://orcid.org/0000-0003-3142-5581
PierrePeterlin
https://orcid.org/0000-0001-5463-6686
MarieC.Béné
https://orcid.org/0000-0002-6569-7414
PEERREVIEW
Thepeerreviewhistoryforthisarticleisavailable athttps://publons.
com/publon/10.1002/hon.3006.
REFERENCES
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s41591‐021‐01542‐z
2.Maillard A,RedjoulR,Klemencie M,etal.Antibody response after
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matopoietic celltransplant recipients. Blood.2021:blood.202
1014232.
3.BaileyAJM,Kirkham AM,Monaghan M,etal.Aportrait of
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Oncol.2022;29(1):337‐349. https://doi.org/10.1016/s2666‐6367
(22)00650‐9
4.ZhaoX,LiD,RuanW,etal.Effectsofaprolonged boosterinterval
onneutralization ofomicron variant.NEnglJMed.
2022;386 (9):894‐896. https://doi.org/10.1056/NEJMc2119426
5.PajonR,Doria‐Rose NA,ShenX,etal.SARS‐CoV‐2 omicron
variantneutralization aftermRNA‐1273 booster vaccination. N
EnglJMed.2022;386 (11):1088‐1091. https://doi.org/10.1056/
NEJMc2119912LETTER TOTHEEDITOR
/3 | a1493134f77670cb875d31694d924d9a52488db1 | page_0002 |
Retrospective Clinical Research Report
Adherence to supplemental
vitamin D intake and infant
weight gain: a retrospectivecohort study in ruralsouthwestern China
Xiaofei Zheng1,*, Qijun Wu2,*, Dadong Weng3,
Yeju Fu3, Duxian Yue3and Yu Wang1
Abstract
Objectives: We tested the hypothesis that vitamin D supplementation is associated with early
body mass index (BMI) patterns.
Methods: Using retrospective longitudinal data of infants and toddlers in a 2-year follow-up
study, we identified BMI-for-age Z score (BAZ) growth trajectories using latent class growth
modeling (LCGM) analysis. Logistic regression models were adopted to investigate the impact of
vitamin D supplementation on BAZ patterns.Results: Five BAZ trajectories were derived using LCGM. Higher intake of vitamin D supplement
was significantly associated with lower odds of rapid weight gain or mild undernutrition in the first 2
years (odds ratios: 0.60–0.83 for class 2: early rapid growth; 0.69–0.82 for class 3: early weight loss,late rapid growth; 0.83–0.89 for class 4: suboptimal growth). Adherence to 400 IU Vitamin D /C211
year was significantly associated with less likelihood of being in class 3 (odds ratio 0.17).
Conclusions: Among infants and toddlers aged 0 to 2 years, dietary supplementation of vitamin
D is insufficient in rural areas of China. Insufficient vitamin D intake was found to be associated
with suboptimal BAZ growth at early stages. Effective collaboration is needed between pedia-
tricians and caregivers, to assure adherence to the nutritional guideline.
Keywords
Vitamin D, infant, growth, poverty, China, body mass index
Date received: 23 July 2019; accepted: 2 October 2020
1Department of Child Health Care, Shanghai Children’s
Hospital, Shanghai Jiao T ong University, Shanghai, China
2Zhenxiong People’s Hospital, Zhaotong, Yunnan, China
3Wude Health Center, Wude, Zhenxiong, Zhaotong,
Yunnan, China*These authors contributed equally to this work.
Corresponding author:
Yu Wang, Department of Child Health Care, ShanghaiChildren’s Hospital, Shanghai Jiao T ong University, Luding
Road 355, Putuo District 200062, Shanghai, China.
Email: [email protected] of International Medical Research
48(11) 1–13
!The Author(s) 2020
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Introduction
The incidence of childhood obesity in China
has increased dramatically in recent years,which has become a public health threat forChinese children,
1as in many low- and
middle- income countries confronted withsimilar challenges.
2,3Surveillance data
reveal that in 2015, China had the highestnumber of obese children (15.3 million)worldwide.
4Although considerable atten-
tion has been given to addressing this
issue, national progress toward preventing
childhood obesity remains unbalanced. Theproblem is more prominent in urban areasbut the prevalence of overweight and obe-sity in rural areas has caught up quicklyover the past decade.
5Growth acceleration
during infancy, mainly driven by inappro-priate parental feeding and behaviors, hasbeen found to increase the susceptibility tobeing overweight and obese among youngschool-age children in rural western China.
6
Rapid gain in the body mass index (BMI)during early stages of growth (0–2 years)increases subsequent obesity risk.
7,8Nearly
half of children who are overweight at age 2years may develop obesity or remain over-weight in adolescence,
9which are both
linked to a series of health risks in adult-hood.
10Therefore, interventions to prevent
childhood obesity should focus on the first1000 days of life, that is, from before birthto age 2 years.
11
Vitamin D is an indispensable micronu-
trient that has numerous biological effectson children’s growth and development.
12
There is strong consensus that infantsshould receive daily vitamin D supplemen-tation during the first year of life, toestablish a healthy vitamin D status.
13,14
Moreover, there is growing concern overthe association between vitamin D andpediatric anthropometric status, consider-ing that adipose tissue has been identifiedas an extraskeletal target of the vitamin D
endocrine system.
15Cross-sectional studieshave confirmed that sufficient vitamin D
concentrations among children and
adolescents are associated with lower risksof cardiovascular disease and metabolic
syndrome.
16,17By contrast, an inverse sig-
nificant correlation between low plasma25-(OH)vitamin D (25OHD) and obesity
has also been reported.
18–20However, it
remains unclear whether vitamin D reple-tion influences childhood BMI-for-age
Z score (BAZ) patterns at young ages,
which could be important because earlygrowth during the first 2 years of life pre-
dicts the prevalence of obesity and other
noncommunicable diseases (NCDs) later
in the life course.
21,22
In this paper, we aimed to assess the
hypothesis that 1) the dosage of vitamin D
supplementation is associated with early
BAZ growth patterns; 2) high adherenceto the recommended dietary allowance
(RDA) of daily vitamin D intake (400 IU/
day) for at least 1 year is associated withoptimal BAZ trajectories, and low adher-
ence could lead to unsatisfactory outcomes.
Methods
Study design and participants
The study was conducted in a low-income
rural area of southwestern China, Zhenxiong
County, Zhaotong City, Yunnan Province.23
This area is geographically located on the
Yunnan–Guizhou Plateau on the Chinese
mainland at 630 to 2416 m above sea level.
A series of observational retrospective
longitudinal data was generated from rou-
tine physical examination of children who
were registered at birth and followed upduring 0 to 24 months in the Health
Center of Wude Town, Zhen Xiong
County, Zhaotong City, Yunnan Province.This center was selected as it is a collabora-
tive institution of Shanghai Children’s
Hospital affiliated to Shanghai Jiao Tong
University, and members of the research2 Journal of International Medical Research | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0001 |
team are active volunteer pediatricians at
this center. This study received exemptionfrom an ethics review by the ResearchEthics Commission of Zhen Xiong
People’s Hospital because the study
involved the use of existing data. Therequirement for participants’ signedinformed consent was waived as the datawere de-identified and all identifiable pri-vate information of participants could notbe ascertained by any means.
Anthropometric measurements and
nutritional assessment
For anthropometric measurements, one
research assistant participated on the physi-cal examination team of Wude HealthCenter between May 2016 and June 2018.
All parents were informed about the physi-
cal examinations via notification from thelocal villagers’ office. An electronic lengthand weight scale was used to assess therecumbent length and weight of children,measured by well-trained professionalsfollowing the guidance of the WorldHealth Organization.
24Measurements were
obtained with children wearing light clothingand no shoes. Weight was measured to thenearest 0.1 kg and length to the nearest 0.1cm. The raw measurements of length and
weight were converted to weight-for-age
Z-score (WAZ) and BAZ using the WorldHealth Organization sex- and age-specificreference standards (WHO MulticenterGrowth Reference Study Group 2006
25).
Vitamin D supplementation and
covariates
The primary variable of interest was infant
daily vitamin D intake. At each health visitduring the first 2 years, this was measuredby questioning mothers in face-to-faceinterviews, as follows: “At what dosageand for how long has your child been sup-
plemented with vitamin D (prescribedvitamin D and any vitamin D sup-
plements)?” Supplementation for each
infant was clearly recorded; data of
brands were not collected. The daily use
was calculated by considering both dose
and frequency.
26For example, if an infant
consumed 400 IU vitamin D every day, thedaily use would be 400 IU; if the dosage was
400 IU every other day, the daily dose was
considered to be 200 IU/day. We also
included covariates of sex, gestational age
(weeks), birth weight Z-score,
27maternal
age at delivery (years),28maternal educa-
tion attainment29(<12 years, illiterate to
primary school graduate; /C2112 years,
middle school graduate and over), season
of birth,30whether exclusively breastfed
during the first 6 months,2and duration
of daily outdoor activity (hours).31
Analytical procedure
Data pre-processing. Among 2100 newborns
registered in the system, 1233 infants who
were followed up had complete data on
anthropometry and vitamin D use atbirth, 3 months (12–16 weeks), 6 months
(24–28 weeks), 1 year (10–14 months),
1.5 years (16–20 months), and 2 years (22–
26 months). Selection of these age time
points was designed to create a more robust
growth curve at similar follow-up times
using comparable anthropometric data.
32
We excluded 51 preterm (gestational age[GA]<37 weeks) and post-term (GA /C2142
weeks) infants from the analysis because
the WHO growth trajectories were not appli-
cable to these infants.
33Infants with unrea-
sonable BAZ ( </C05o rW A Z >5) at any of
the abovementioned time points were
excluded (n ¼23) owing to potentially incor-
rect data entry.
Growth patterns and grouping. To derive dif-
ferent BAZ growth patterns, we performed
a latent class growth modeling (LCGM)
analysis, a technique designed to identifyZheng et al. 3 | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0002 |
homogeneous subgroups within a heteroge-
neous population34that is very applicable
to the study of longitudinal developmental
trajectories at early stages.7,32The number
of BAZ growth profiles was decided on the
basis of the Bayesian information criterion
(BIC); models with smaller values of the
BIC imply a better fit.
Statistical analysis. Standardized BAZ and
WAZ at birth were estimated using the pub-
licly available software, WHO Anthro 3.2
(https://www.who.int/growthref/tools/en/).
Other statistical analysis was carried out
using RStudio (The R Project for Statistical
Computing, Vienna, Austria). LCGM analy-
sis was performed using the “poLCA” pack-
age in RStudio.35Frequencies with
percentage or means with standard deviation
(SD) of basic characteristics were calculated,
as descriptive statistics. Logistic regression
models were performed to estimate the effects
of vitamin D use on BAZ growth when cova-
riates were controlled. Relationships between
daily supplementation with 400 IU /C211y e a r
and BAZ growth patterns in early childhood
were also assessed in the logistic regression
model, where the dosage of vitamin D was
divided by 100.
Results
In total, 1159 eligible infants and toddlers
were included in the final analysis. The
characteristics of the study population are
summarized in Table 1. Boys accounted for
56.6% of the final sample. The mean age of
gestation was 39.8 weeks, with an average
birthweight Z-score /C00.2.
Growth trajectories in the first 2 years
Using LCGM, the BIC outputs for the
fitted models from two to six growth cate-
gories were 18,601.46, 18,116.95, 17,981.84,17,946.97, 18,001.04. Therefore, the BAZgrowth of the study population was divided
into five categories, as depicted in Figure 1.
Trajectory class 1, comprising 42.4% of
the study population, was termed “normativegrowth”. These children had mean estimatedBAZ/C00.20 at birth and showed an optimal
normative growth pattern around standardlevels. BAZ growth class 2, denoted “earlyrapid growth”, comprised 10.9% of children.This trajectory class showed a rapid weightgain early, within the first 3 months of life,and remained 1 unit above the ideal growthstandards until 2 years of age. BAZ growth
trajectory class 3 (8.9%), “early weight loss,
late rapid growth”, included children whoexperienced weight loss during the first 3months but who were substantially accelerat-e db y2S Dw i t h i nt h ef o l l o w i n g2y e a r s .T h eBAZ trajectory class 4 comprised childrenwith unsatisfactory growth, close to mildmalnutrition status,
36which continued to
decline over time to 1 SD below the standardlevel until age 2 years; this class was termed“suboptimal growth” and comprised 12.5%
of the study population. The trajectory class
5, “early normative, late rapid growth”,showed a pattern similar to normativegrowth in the first 6 months but then deviatedbyþ1 SD above standard growth; this class
comprised 21.9% of the whole sample.
Using analysis of variance or chi-square
tests, some disparities in the distribution ofdemographic characteristics were observedamong the different groups. Therefore, theheterogeneity of participant characteristicswas accounted for by adjusting for poten-tial confounders (Table 1). The table alsopresents the difference between includedand excluded term newborns in this study,in which most characteristics were not sig-
nificantly different at baseline.
Status of supplemental vitamin D use
Data on the proportion of children
who were ever given supplemental vitaminD at each visit are shown in Figure 2.4 Journal of International Medical Research | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0003 |
Table 1. Characteristics of study participants identified using latent class growth modeling: Zhenxiong, Zhaotong, Yunnan, China 2016–2018 (n ¼1159).
VariablesClass 1
(n¼491)Class 2
(n¼127)Class 3
(n¼103)Class 4
(n¼145)Class 5
(n¼293)T erm infants,
included(n¼1159)T erm infants,
excluded(n¼904) P value
5P value6
Boys (vs. girls)1289 (58.9) 44 (34.6) 84 (81.6) 98 (67.6) 141 (48.1) 656 (56.6) 492 (54.4) <0.001 0.303
Gestational age (weeks)239.8 (0.6) 39.8 (0.7) 39.7 (0.7) 39.8 (0.6) 39.8 (0.7) 39.8 (0.7) 39.5 (0.8) 0.384 <0.001
Birthweight Z-score2,3/C00.2 (0.5) 0.2 (0.7) /C00.5 (0.5) /C00.4 (0.5) /C00.1 (0.6) /C00.2 (0.6) /C00.2 (0.7) 0.142 0.940
Maternal age at delivery (years)226.6 (4.6) 27.0 (4.8) 26.2 (4.2) 26.9 (4.2) 26.7 (4.32) 26.7 (4.4) 12.6 (5.63) 0.891 0.008
Maternal higher education2147 (29.9) 32 (25.2) 10 (9.7) 13 (9.0) 58 (19.8) 260 (22.4) 197 (21.8) <0.001 0.764
EBF in first 6 months
(EBF vs. non-EBF)2,4240 (48.9) 79 (62.2) 52 (50.5) 77 (53.1) 144 (49.1) 592 (51.1) 438 (48.5) 0.094 0.254
Season of birth1
Spring (Mar to May) 112 (22.8) 23 (18.1) 23 (22.3) 35 (24.1) 77 (26.3) 270 (23.3) 219 (24.2) 0.299 0.071
Summer (Jun to Aug) 92 (18.7) 31 (24.4) 16 (15.5) 29 (20.0) 71 (24.2) 239 (20.6) 223 (24.7)Autumn (Sep to Nov) 143 (29.1) 33 (26.0) 36 (35.0) 36 (24.8) 75 (25.6) 323 (27.) 233 (25.8)Winter (Dec to Feb) 144 (29.3) 40 (31.5) 28 (27.2) 45 (31.0) 70 (23.9) 327 (28.2) 229 (25.3)
1Chi-square tests were performed to determine the P value for categorical variables. Results are expressed as number (percentage).
2Analysis of variance was adopted to determine the P value for continuous variables. Results are expressed as mean (standard deviation).
3Z-scores were calculated according to age- and sex-specific World Health Organization child growth standards.
4Maternal higher education was defined as completing >12 years education.
5Comparison among growth patterns.
6Comparison between term infants included and excluded in the analysis.
EBF , exclusively breastfed.Zheng et al. 5 | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0004 |
Overall, the highest proportion of children
routinely receiving supplemental vitamin D
at most visits were those with normativegrowth (trajectory class 1). By contrast,fewer than 25% of children in class 3
(early weight loss, late rapid growth) ever
received vitamin D within the first 2 yearsof life. In terms of adherence to the
Figure 1. Five BAZ growth patterns of infants and toddlers in the first 2 years according to LCGM
Trajectory class 1: normative growth (n ¼491); Trajectory class 2: early rapid growth (n ¼127); Trajectory
class 3: early weight loss, late rapid growth (n ¼103); Trajectory class 4: suboptimal growth (n ¼145);
Trajectory class 5: early normative, late rapid growth (n ¼293).
BAZ, body mass index (BMI)-for-age Z score; LCGM, latent class growth modeling.
Figure 2. Proportion of children who received supplemental vitamin D at each visit.6 Journal of International Medical Research | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0005 |
recommendations, in general, only 37.1% of
the study population received supplementalvitamin D /C211 year. As displayed in Figure
3, class 1 (normative growth) accounted for
the highest percentage (41.8%) of partici-pants who received at least 400 IU vitaminD supplementation daily for the first year.
Significant differences were found when
comparing class 1 with classes 3 (earlyweight loss, late rapid growth, p <0.001)
and 4 (suboptimal growth, p ¼0.003).
Vitamin D supplementation and growth
trajectories
We analyzed the association between daily
vitamin D intake dosages and BAZ growthpatterns, as shown in Table 2. In compari-
son with those who had normative growth
(class 1), children in classes 2 ,3, and 4 had,respectively, 0.60 to 0.83 (class 2), 0.69 to0.82 (class 3) and 0.83 to 0.89 (class 4) times
the risk of having early or late rapid weight
gain with every 100 IU/day vitamin Dincrease, suggesting that vitamin D supple-
mentation had a protective effect against
rapid growth.
The odds ratios (ORs) of the covariate
models are presented in Table 3, which
demonstrated that 400 IU vitamin D dailyfor<1 year (as compared with adherence to400 IU vitamin D per day /C211 year) was
related to the type of BAZ growth patternthat a child had the greatest likelihood of
developing. Compared with reference class
1, infants who received daily 400 IU for /C211
year showed 0.17 greater odds of develop-ing rapid weight gain prior to 2 years of
age (class 3). The association remained
significant (OR 0.17, 95% CI 0.04–0.50,p<0.001) after accounting for all relevant
covariates (model 2). A similar association
was found for class 4, which indicated that
infants with maternal adherence to the rec-ommendations had a 0.53-fold decreasedlikelihood of suboptimal growth (OR 0.53,
95% CI 0.35 /C00.80, p ¼0.003), even
though this association was attenuatedwhen the covariates were fully adjusted. Asimilar association was found for class 4,
which indicated that infants with maternal
adherence to the recommendations had a0.53-fold decreased likelihood of subopti-mal growth, even though this association
was attenuated when the covariates were
fully adjusted.
Discussion
The main findings of our study were thatadditional vitamin D intake in infancy hasbenefit in the development of optimal BAZ
growth, and low or no vitamin D supple-
mentation is more likely to be associatedwith poor growth outcomes during thefirst 2 years of life. Our results lend support
to the recommendation that adherence to
the RDA of 400 IU/day vitamin D for 1year increases the likelihood of a bettergrowth trajectory in comparison to supple-
mentation for less than a year. Although
the benefits of vitamin D with respect tocalcium and bone homeostasis have beenadequately demonstrated, little evidence as
to the effects of vitamin D on BAZ growth
for infants and toddlers during the first1000 days is available. We believe that ourresults will serve as a basis for further
Figure 3. Proportion of children who received at
least 400 IU vitamin D daily for 1 year in each group
***P<0.001; **P <0.01.Zheng et al. 7 | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0006 |
Table 2. Logistic regression for daily dose vitamin D (unit: 100 IU) according to growth trajectory at all visits: Zhenxiong, Zhaotong, Yunnan, China 2016–
2018 (n ¼1159).
Age at visit1 months
OR (95% CI)3 months
OR (95% CI)6 months
OR (95% CI)8 months
OR (95% CI)12 months
OR (95% CI)18 months
OR (95% CI)24 months
OR (95% CI)
Class 1 (Reference)
Class 2
Unadjusted 0.89 (0.80–0.99) 0.90 (0.81 /C01.00) 0.84 (0.75 /C00.94) 0.82 (0.73 /C00.92) 0.81 (0.72 /C00.9) 0.80 (0.72 /C00.89) 0.78 (0.69 /C00.88)
Adjusted10.83 (0.72 /C00.95) 0.82 (0.72 /C00.94) 0.79 (0.68 /C00.91) 0.79 (0.68 /C00.91) 0.73 (0.61 /C00.85) 0.70 (0.57 /C00.84) 0.60 (0.45 /C00.76)
Class 3
Unadjusted 0.74 (0.65 /C00.83) 0.72 (0.63 /C00.81) 0.67 (0.59 /C00.77) 0.70 (0.61 /C00.79) 0.71 (0.62 /C00.81) 0.74 (0.65 /C00.84) 0.72 (0.62 /C00.82)
Adjusted 0.82 (0.70 /C00.96) 0.78 (0.66 /C00.91) 0.69 (0.58 /C00.82) 0.73 (0.62 /C00.86) 0.75 (0.63 /C00.88) 0.80 (0.68 /C00.94) 0.78 (0.64 /C00.92)
Class 4
Unadjusted 0.83 (0.75 /C00.92) 1.05 (0.95 /C01.15) 0.93 (0.84 /C01.04) 0.93 (0.84 /C01.03) 0.97 (0.88 /C01.07) 0.89 (0.80 /C00.97) 0.90 (0.82 /C00.99)
Adjusted 0.96 (0.85 /C01.08) 1.28 (1.14 /C01.43) 1.11 (0.99 /C01.25) 1.10 (0.98 /C01.24) 1.14 (1.02 /C01.27) 1.04 (0.93 /C01.16) 1.09 (0.97 /C01.23)
Class 5
Unadjusted 0.90 (0.84 /C00.98) 0.91 (0.84 /C00.98) 0.82 (0.75 /C00.89) 0.84 (0.77 /C00.91) 0.91 (0.84 /C00.98) 0.86 (0.79 /C00.92) 0.86 (0.79 /C00.93)
Adjusted 0.94 (0.85 /C01.02) 0.93 (0.85 /C01.02) 0.83 (0.75 /C00.92) 0.87 (0.79 /C00.96) 0.96 (0.88 /C01.05) 0.89 (0.80 /C00.97) 0.89 (0.80 /C00.98)
1Adjusted for sex, gestational age (weeks), birthweight Z-score, maternal age at delivery (years), maternal education /C2112 years, exclusively breastfed in first 6 months,
duration of daily outdoor activity (hours).
OR, odds ratio; CI, confidence interval.8 Journal of International Medical Research | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0007 |
Table 3. Association of daily vitamin D supplementation /C2112 months with growth patterns in the first 2 years: Zhenxiong, Zhaotong, Yunnan, China
2016–2018 (n ¼1159).
Model 1 Model 2 Model 1 Model 2
OR (95% CI) OR (95% CI) OR (95% CI) OR (95% CI)
Class 1 (Reference)
Class 2 Class 4
Vitamin D supplementation /C2112 months 0.92 (0.60 /C01.41) 1.23 (0.63 /C02.31) 0.53 (0.35 /C00.80) 1.32 (0.76 /C02.23)
Boys (vs. girls) 0.37 (0.24 /C00.57) 0.35 (0.22 /C00.54) 1.47 (0.99 /C02.21) 1.41 (0.94 /C02.13)
Gestational age (weeks) 0.82 (0.60 /C01.13) 2.60 (1.87 /C03.64) 0.96 (0.72 /C01.30) 0.52 (0.36 /C00.74)
Birthweight Z-score 2.79 (2.02 /C03.90) 0.88 (0.64 /C01.21) 0.56 (0.40 /C00.79) 1.05 (0.78 /C01.43)
Maternal age at delivery (years) — 1.00 (0.96 /C01.05) — 1.02 (0.98 /C01.07)
Maternal education /C2112 years — 0.60 (0.28 /C01.27) — 0.18 (0.08 /C00.38)
Exclusively breastfed in first 6 months — 1.49 (0.96 /C02.34) — 1.07 (0.71 /C01.60)
Daily outdoor activity (hours) — 0.67 (0.51 /C00.86) — 0.98 (0.79 /C01.22)
Class 3 Class 5
Vitamin D supplementation /C2112 months 0.19 (0.10 /C00.34) 0.17 (0.04 /C00.50) 1.00 (0.74 /C01.35) 1.98 (1.30 /C03.02)
Boys (vs. girls) 3.50 (2.04 /C06.30) 3.43 (1.98 /C06.25) 0.65 (0.49 /C00.87) 0.65 (0.48 /C00.87)
Gestational age (weeks) 0.98 (0.71 /C01.37) 0.31 (0.20 /C00.48) 0.92 (0.74 /C01.13) 1.06 (0.82 /C01.37)
Birthweight Z-score 0.34 (0.22 /C00.52) 1.03 (0.74 /C01.44) 1.15 (0.89 /C01.47) 1.02 (0.82 /C01.27)
Maternal age at delivery (years) — 0.98 (0.92 /C01.03) — 1.01 (0.98 /C01.05)
Maternal education /C2112 years — 0.96 (0.27 /C04.59) — 0.30 (0.18 /C00.49)
Exclusively breastfed in first 6 months — 0.96 (0.58 /C01.56) — 0.88 (0.64 /C01.19)
Daily outdoor activity (hours) — 0.61 (0.46 /C00.80) — 0.80 (0.67 /C00.95)Zheng et al. 9 | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0008 |
studies on the vital clinical importance of
early micronutrient supplementation for
body growth and the prevention of obesity
later in life. Our findings also highlight the
importance of collaboration between pedia-
tricians and caregivers in implementingnutritional guidelines in developing coun-
tries, particularly in rural areas.
We note that at most age time points,
infants with higher vitamin D intakes were
more likely to have a normative BAZ
growth pattern. In other words, relativelylow doses or no use of vitamin D was asso-
ciated with the risk of excess BAZ growth.
To our knowledge, limited longitudinalstudies exist regarding the association
between vitamin D intake and infant BAZ
patterns. A Polish cross-sectional studyreported that a greater percentage of chil-
dren aged 1 to 3 years did not achieve ade-
quate vitamin D intake among obesechildren than normal weight children.
37
Weker et al. also detected an insufficientsupply of vitamin D among overweightand obese toddlers.
38Conversely, vitamin
D administered to children was not linked
to anthropometric benefits in a trial con-ducted during pre-puberty,
39when the
demand for nutrients for rapid growth is
not high as in infancy. Clearly, these findingsrequire replication in other cohort studies.
Nutritional intake and ultraviolet B
radiation-induced synthesis in the skin areboth natural sources of human vitamin D.
However, few foods contain vitamin D
40
and infants, whose skin is thinner, are
more vulnerable to DNA damage caused
by exposure to direct sunlight.41Moreover,
attaining adequate 25OHD concentrations ischallenging for children residing at high alti-
tudes,
42,43such as the participants in our
study, which highlights the need for addi-tional intake despite adequate sunshine.
Vitamin D supplementation has been
shown to generally protect against low25OHD in systematic reviews and meta-
analyses.
44,45Similar to other scientific orgovernmental nutritional guidelines, e.g.,
the Institute of Medicine46and guidelines
in the United States47and Canada,48a
dose of 400 IU per day vitamin D is recom-
mended from birth until age 12 months by
the Chinese Nutrition Society. Nevertheless,we found that only a few parents or care-
givers in this study had adhered to this rec-
ommendation. Surprisingly, over one-thirdof infants received no vitamin D supplemen-
tation in the first year, and the percentage of
toddlers who received vitamin D supple-ments decreased with increased age.
Pediatricians play a critical role in ensuring
that infants receive adequate amounts ofvitamin D because advice from these health
care professionals is considered more reliable
among child care providers.
49In rural areas,
where mothers have lower education levels
and limited access to nutrition-related
knowledge, pediatricians hold an importantposition in protecting child health.
Among all covariates, we noted that chil-
dren with longer durations of outdooractivity were less likely to have rapid
weight gain within the first 2 years. This
may be attributable to the benefits of sun-shine exposure, leading to vitamin D syn-
thesis.
50Maternal education more than 12
years was also a protective factor that hadimpact on BAZ growth in this low-income
area, as in studies conducted in other devel-
oping parts of the world.
51
There are some limitations in the study.
Nearly half of the registered newborns were
unavailable for follow-up at all time points,largely owing to migration losses, which
might cause potential selection bias in rela-
tion to vitamin D supplementation. Use ofvitamin D was self-reported by caregivers
and could possibly result in social desirabil-
ity bias and recall bias regarding actual feed-ing behavior. Under the conditions at local
health centers, biomarkers such as 25OHD
were unable to be measured. As a commonproblem in observational research, we
could not consider all possible covariates.10 Journal of International Medical Research | 8857b3e80c4283e7d81b1c1228de1a9b44a88fbd | page_0009 |
For example, we did not include factors
related to maternal nutrition and cutaneoussynthesis of vitamin D, e.g., habitual cloth-
ing and skin pigmentation.
Conclusions
Among infants and toddlers aged 0 to 2years, sufficient dietary supplementation
with vitamin D is lacking in rural areas ofChina. Insufficient vitamin D intake wasfound to be associated with suboptimal
BAZ growth at early stages. Vitamin D
intake of at least 400 IU/day, sustained forthe first 1 year of life, resulted in improvedbody growth within 0 to 2 years. Effective
collaboration between pediatricians and
caregivers is needed, to assure adherence tothe nutritional guideline.
Acknowledgements
The authors thank the study participants, their
parents, and doctors at Wude Health Center, as
well as the researchers in this study.
Declaration of conflicting interest
The authors declare that there is no conflict of
interest.
Funding
This study was supported by the Shanghai
Municipal Committee of Science and Technology
(grant numbers: 16411970100, 18411967700);Shanghai Children’s Health Services Capacity
Planning Special Program for Advanced
Pediatric Overseas Research Team Training
Program (grant number: GDEK201710); and
Shanghai Municipal Health Commission (grantnumber: GWV-10.1-XK19).
ORCID iD
Yu Wang https://orcid.org/0000-0001-6212-
4816
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Slowing DNA Translocation in a Solid State Nanopore
Daniel Fologea ,
Department of Physics, University of Arkansas, Fayetteville, AR72701
James Uplinger ,
Department of Physics, University of Arkansas, Fayetteville, AR72701
Brian Thomas ,
Department of Physics, University of Arkansas, Fayetteville, AR72701
David S. McNabb , and
Department of Biology, University of Arkansas, Fayetteville, AR72701
Jiali Li*
Department of Physics, University of Arkansas, Fayetteville, AR72701
Reducing a DNA molecule’s translocation speed in a solid-state nanopore is a key step
towards rapid single molecule identification. Here we demonstrate that DNA translocation
speeds can be reduced by an order of magnitude over previous results. By controlling the
electrolyte temperature, salt concentration, viscosity and the electrical bias voltage across
the nanopore we obtain a 3 base/microsecond translocation speed for 3 kilobase double-
stranded DNA in a 4–8 nm diameter silicon nitride pore. Our results also indicate that the
ionic conductivity inside such a nanopore is smaller than it is in bulk.
A nanopore based sensor can detect single DNA molecules, and nanopore sensing represents
a potential future technology for rapid DNA sequencing. Since Kasianowicz et al 1
demonstrated that individual DNA molecules could be electrophoretically driven through a
single ~2 nm diameter alpha-hemolysin protein nanopore, several studies have clarified and
extended the utility of this nanopore 2–6. Recently, solid state 7–14 nanopores have also been
used to detect DNA molecules. The DNA nanopore translocation process has also been
investigated theoretically 15–19. Several serious technically problems remain to be solved if
the goal of rapid molecule characterization and sequencing is to be achieved in solid state
nanopores. One is that the measured DNA translocation speed of ~30 bases/µsec requires
electronic sensing system at extremely high bandwidth, and the concomitant electronic noise
poses serious limitations in electrically discriminating between bases. Below we
demonstrate how the bandwidth requirements can be reduced by an order of magnitude by
slowing down the molecule translocation speed.
The detection of a DNA molecule is performed by placing a nanopore chip between two
separated chambers, electrically connected only by an ionic solution inside the nanopore.
When a voltage is applied, a negatively charged DNA molecule in the vicinity of the
nanopore will be captured by the electric field, and forced to pass through the nanopore from
the negative (Cis) side to the positive (Trans) side. A molecule inside the nanopore causes a
detectable ionic current blockade. Both the translocation time (dwell time, t d) and the
amplitude of the blockade (current drop, ΔIb) are dependent on the solution conditions (ionic
concentration, viscosity, and temperature), properties of the nanopore, bias voltage and the
passing molecule. DNA translocation is a very complex process, but it can be envisioned as
*Corresponding author. [email protected].
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resulting from a balance between the electric driving force and viscous drag. The nanopore
electrical behavior in ionic solution seems to be an ohmic one, with the electrical current
blockages proportional to the applied voltage14:
(1)
where σ is the solution conductivity, V the applied voltage across the nanopore, H the
effective thickness of the nanopore, and A DNA denotes the hydrodynamic cross section of
the translocating molecule. Using a simple equation of force balance between the electric
force in the nanopore and the viscous drag over the whole molecule one finds for the
translocation time 7
(2)
where η is the viscosity of the solution, λ and L DNA are the linear charge density and length
of the DNA molecule, respectively, and K is a constant of proportionality accounting for
complex issues beyond the capabilities of the simple model. Equations (1) and (2) are
coupled by the fact that σ will depend on η (σ ~1/η) 20. In addition, σ, η and λ will depend
on the temperature and the concentration of ions in the nanopore.
In this work we explore the various accessible experimental factors in equations (1) and (2)
for slowing DNA molecule transport through nanopores, namely viscosity, bias voltage, salt
concentration, and temperature. Our nanopores are fabricated in a free standing 280 nm
thick silicone nitride membrane supported by a 380 µm thick silicone substrate using a
combination of Focus Ion Beam milling and feedback controlled ion beam sculpting 8,21.
Due to the fabrication process, individual nanopores may have different thickness, diameter,
shape or even surface charge in solution, all of which may result in various translocation
characteristics. In order to avoid these problems, a single nanopore with a diameter of 4–8
nm was used for each experiment described below. All measurements are performed in a
typical TE (10 mM Tris, 1 mM EDTA) buffer (pH=7.5), with different concentrations of
KCl (1–3 M) and/or different concentrations of glycerol (0–50%). A linear 3 kbp plasmid
(pSP65) was added to our Cis chamber in a 10nM final concentration. Ionic current signal
through solid state nanopores was measured and recorded using an integrated Axopatch
200B patch-clamp amplifier system (Axon Instrument) in resistive feedback mode. The 10
kHz low pass Bessel filter in the Axopatch 200B was selected for all measurements in this
work. At this setting, the whole measuring system was tested and calibrated with artificial
current blockages, ideal square pulses, generated from a function generator (Agilent
33250A). The pulse width generated was 20, 40, 60, 80, 100, 140, 160 µsec, and the pulse
height was about 100 pA. The recorded data was analyzed with the same MatLab routines
for real DNA translocation. When the pulse width is less than 100 µsec, the pulse height will
be attenuated, but the time durations (the width of half height) remain correct. When the
time duration measured in this work was less than 100 µsec, the current blockage amplitude
was corrected with this calibration. The current blockages ΔIb and the translocation times t d
are extracted from the recorded data using custom Matlab® routines. Unless other wise
mentioned the bias voltage is set to 120 mV. For every DNA data set the number of
recorded events is between 3,000 and 12,000, and the errors of measurements (ratio between
the standard deviation and the mean) are at most 15% except the low temperature-low
voltage experiment where the error is about 25%.Fologea et al. Page 2
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Viscosity Study
We studied DNA translocation in different bulk solution with viscosities between 1–5.3 cP
(centipoises). The viscosity of KCl-TE solution was changed by adding glycerol (0–50%),
while keeping the KCl concentration constant at 1.5 M, and was measured using a model
GV-2100 (Gilmont Instruments) drop ball viscometer. Plotting the peak positions of current
drops and translocation times as a function of viscosity, our results in Figure 1a show: (1)
the current blockades ( ΔIb, filled squares) decrease inversely proportional to the viscosity,
which can be explained with eq.1 by the fact that conductivity σ is proportional to 1/ η; (2)
the translocation times increase linearly with viscosity, as predicted by eq. 2. The ratio of
open pore current to current drop for all measured viscosities is about 45 ± 5 indicating the
open pore current and current drop are actually modified in the same manner when the
viscosity is changed (data not shown). The most important result from our viscosity study is
that the translocation time can be increased by increasing the solution viscosity. Increasing
the viscosity about 5 times (adding 50% glycerol), increases the translocation time, and thus
the temporal resolution, by about 5 times. Furthermore, the current blockage is still large
enough for signal analysis.
The current blockages versus the translocation times for DNA translocation (the scatter plot)
for 10% (1.3 cP) and 50% (5.3 cP) glycerol are shown in Figure 1b. These scatter plots show
the same pattern as previously reported 7,14 for KCl solution with 0% glycerol: the DNA
appears to exist in two folding states. The first one, characterized by peak positions for
current drops of about 50 pA and translocation times of about 550 µsec for 50% glycerol,
and 120 pA and 140 µsec for 10% glycerol, belongs to unfolded DNA molecule passing the
nanopore. The second more distributed one (the tails of the density plot, Figure 1b) shows an
increased current drop and a reduced translocation time, corresponds to folded DNA
molecules.
Bias Voltage Study
Figure 2 shows the voltage dependency of DNA translocation through a ~6nm silicon nitride
pore. The measured current blockages are linear with applied voltage in 20–100 mV range,
and the translocation times are inversely proportional to the voltage. Although at the lowest
voltage used (20mV) the current drop is very small (about 22 pA), however, the DNA
translocation signal can still be easily measured and analyzed.
These results are consistent with previously reported values for solid state nanopores 7,14
and for the alpha-hemolysin 1,2,22,23 protein pores showing an inverse relationship between
translocation time and applied voltage, however, they are in contradiction with results
obtained for smaller diameter pores11. For an alpha-hemolysin nanopore, it has been
suggested that there exist an energetic barrier that DNA needs to overcome before
translocation can occur 2,22,23. The height of this barrier is determined by both electrostatic
interactions and geometrical restrictions in a confined volume 23,24. DNA passes the protein
pore only for applied voltages higher than 40–60 mV 22,23. In our case, as shown in Figure
2, the DNA molecules pass through our silicon nitride pore with applied voltage as low as
20 mV. This suggests the energy barrier for our silicon nitride pores is lower than that for
the protein pore, probably due to the increased diameter of the nanopore and reduced
electrostatic interactions.
Salt Concentration Study
In order to study the salt concentration dependency of DNA translocation, the KCl
concentration of TE buffer was changed from 1 to 3 M. In this range, the macroscopic
conductivity (measured with a VWR Traceable® Expanded-Range Conductivity Meter) isFologea et al. Page 3
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not linear with respect to KCl concentration, as saturation is observed in the solution’s
conductivity as salt concentration become high (Fig. 3a). When the KCl concentration is
increased from 1 to 3M, the bulk conductivity is increased from 95 to 145 mS/cm. However,
both the open pore current (Fig. 3a, squares) and current blockage (Fig. 3b, squares)
decrease linearly with KCl concentration.
The saturation of the bulk conductivity above 1M KCl can be explained by increased
interionic effects at high KCl concentration, which increases the resistivity of the ionic
solution. The linear relation between the ionic current and KCl concentration in a silicon
nitride nanopore is consistent with recent work done by Stein et al 25, in which the authors
found that the conductance in nanochannels is approximately linear to KCl concentration
between 0.1 and 1M. In this salt concentration range, the ionic current, I, is directly
proportional to ion concentration n, as a limiting case for Levine relationship 26, I ~ n(1+C),
where C is the correction to the bulk conductivity. The fact that both the open pore current
and current drop are linear versus KCl concentration suggests the KCl concentration in a 4–
8 nm silicon nitride nanopore is lower than the bulk, and the mechanism of ions transport
through a nanopore is the same with or without DNA inside the nanopore. The difference
between the conductivity in a nanopore and in bulk is still under investigation.
For the KCl concentration range studied, the translocation time is essentially constant (Fig.
3b), DNA molecules seem to be saturated with counterions so a higher concentration only
slightly increases the translocation time by decreasing the DNA charge (the well known
screening effect of positive ions).
Temperature
We studied the temperature behavior of DNA translocation in TE buffer, containing 1.6 M
KCl and 20% glycerol at 2 different temperatures, 22 °C and 4 °C. The temperature was
kept constant using a HCC-100A (BioScience Tools) temperature controller. In contrast
with the results reported from biological nanopore experiments2,27, which showed a strong
dependency of translocation time related on temperature2,27, at low temperature the DNA
translocation pattern through a silicon nitride nanopore is not strongly modified. When the
temperature was decreased from 22 °C to 4 °C, the current blockage decreased from 210 to
140 pA and the translocation time increased from 165 to 280 µsec (Fig. 4), less than a factor
of two. This behavior supports the supposition of a stronger dependency of protein pore
properties on temperature, not major changes in DNA or bulk transport properties.
Temperature can affect conductivities, mobility, viscosity or the pore channel itself. Again,
we measured the same ratio of open pore current to current drop for different temperatures
which suggests the open pore current and current drop are modified in the same manner by
temperature changes. Also, for our silicon nitride nanopores the transport properties seem to
remain relatively constant, whereas the properties of the bulk such as viscosity increase with
decreasing temperature are the main contributors to the temperature dependence on
translocation time and current drop.
In order to demonstrate the goal of increasing the translocation time, a DNA translocation
experiment was carried out in a 1.6 M KCl-TE solution, containing 20% Gly, at 40 mV and
4° C. Although the recorded current drop was small, about 40 pA, the signal was easily
discernable from noise. The translocation time was about 800 µsec (Fig. 4), almost one
order of magnitude higher than the typical translocation time recorded for a same DNA
molecule passed through a same nanopore at room temperature, bias voltage 120 mV and
1M KCl buffered bulk 7. The first small peak for every histogram (Fig. 4) corresponds to
folded DNA molecules translocating through the nanopore and is characterized by a shorter
translocation time.Fologea et al. Page 4
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The DNA translocation speed through 4–8 nm silicon nitride nanopores is about 30 base/
µsec without glycerol, the same as measured under similar conditions7,14. By adding 50%
glycerol, the translocation speed can be slowed by a factor of 5.5. Decreasing the bias
voltage to ~20mV, slows the DNA translocation speed by a factor of 3, and decreasing the
temperature of our measuring system to 4° C, slows it by a factor of ~2. Combining all of
these factors while maintaining a good signal to noise ratio, the DNA translocation speed
can be slowed by a factor of 10, or an order of magnitude. The strategies used in this work
slow DNA molecules; however, it also slows conducting ions which decreases the current
blockage signal. Our future strategy will be slowing down only the DNA molecules not the
ions.
Acknowledgments
We thank Prof. J. Golovchenko for help of FIB hole preparation and valuable discussions, Dr. W. Oliver for his
helpful comments, B. Ledden for nanopore fabrication, and A. Huang for MatLab program assistance. This work is
supported by NSF/MRSEC 0080054, ABI-111, and NIH1R21HG003290-01.
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25. Stein D, Kruithof M, Dekker C. Phys. Rev. Lett 2004;93:035901. [PubMed: 15323836]Fologea et al. Page 5
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Figure 1.
a) Current blockage ( ■) and translocation time ( ▲) versus viscosity for 3kbp DNA in 1.5 M
KCl-TE solution at 120 mV bias voltage; the solid curves are fits for ΔIb ~1/η and td~ η. b)
The scatter plots for the addition of 10% and 50% glycerol to the ionic solution, the color
scale represents the events density.Fologea et al. Page 7
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Figure 2.
Current blockage ( ■) and translocation time ( ▲) versus applied voltage measured in 1.6 M
KCl-TE buffer containing 20% glycerol. The solid curves represents fits where ΔIb ~V and
td~1/V.Fologea et al. Page 8
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Figure 3.
a) Open current ( ■) (without DNA added) and bulk conductivity ( ▲) change as a function
of salt concentration at120 mV bias voltage. b) Current drop ( ■) and translocation time ( ▲)
change as a function of KCl concentration. The solid curves are linear fits of ΔIb and t d to
KCl concentration.Fologea et al. Page 9
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Figure 4.
Translocation time is increased when the temperature is decreased. At low voltage (40 mV)
and low temperature (4° C) in 1.6 M KCl-TE containing 20% glycerol the translocation time
is about 800 µsec.Fologea et al. Page 10
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A Detergent-Based Assay for the Detection of Promiscuous
Inhibitors
Brian Y Feng and Brian K Shoichet
Department of Pharmaceutical Chemistry, and Graduate Group in Chemistry and Chemical Biology,
University of California-San Francisco, 1700 4th Street, California 94158-2330, USA.
Abstract
At micromolar concentrations, many small molecules self-associate into colloidal aggregates that
non-specifically inhibit enzymes and other proteins. Here we describe a protocol for identifying
aggregate-based inhibitors and distinguishing them from small molecules that inhibit via specific
mechanisms. As a convenient proxy for promiscuous, aggregate-based inhibition, we monitor
inhibition of β-lactamase in the absence and presence of detergent. Inhibition that is attenuated in
the presence of detergent is characteristic of an aggregate-based mechanism. In the 96-well-format
assay described here, about 200 molecules can be tested, in duplicate, per hour for detergent-
dependent sensitivity. Furthermore, we also describe simple experiments that can offer additional
confirmation of aggregate-based inhibition.
INTRODUCTION
Small molecules that specifically inhibit enzymes or modulate protein function are intensely
sought. The dominant technique for discovering such ligands is high-throughput screening
(HTS). In HTS, large libraries of small molecules are assayed for modulation of a target.
Although this technique has had important successes, it is plagued by false-positive ligands.
These artifactual ‘hits’ can outnumber the true inhibitors in a HTS ‘hit list’. Several
mechanisms have been proposed to explain the prevalence of these artifactual hits, including
oxidation potential1, chemical reactivity2 and spectral properties that interfere with assay
readout3. One of the more common mechanisms underlying false-positive inhibition is the
formation of colloidal aggregates through the self-association of organic molecules in aqueous
solutions ( Fig. 1 )4-9. These aggregates typically form at micromolar concentrations and are
often several hundred nanometers in diameter. Once formed, they sequester proteins and non-
specifically inhibit their activity. A wide range of molecules behave this way at screening-
relevant concentrations, including Lipinski-compliant members of screening libraries, bona
fide leads for drug discovery and even drugs6,8,9. If the number of different types of molecules
that can behave in this way is surprising, so too is the actual number of molecules that do so.
At 30 μM, up to 19% of ‘drug-like’ molecules can form aggregates. At 5 μM, about 1-2% of
‘drug-like’ molecules seem to behave in this way, which is still a large percentage considering
that many HTS campaigns aim for a hit rate of less than 1% (ref. 5). Here we describe a counter-
screen for aggregation that can be deployed on a library-wide level, as well as a checklist of
experiments that can be used to confirm whether individual inhibitors are acting through an
aggregation-based mechanism (see ANTICIPATED RESULTS).
Several alternatives to this assay exist, among which are direct physical measurement of
aggregation by dynamic light-scattering (DLS) and apparent solubility by nephelometry.
Correspondence should be addressed to B.K.S. ([email protected])..
COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests.
NIH Public Access
Author Manuscript
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Published in final edited form as:
Nat Protoc . 2006 ; 1(2): 550±553.
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Although it is more direct, we found DLS to be both time-consuming and harder to interpret,
owing to problems with signal-to-noise in the plate-based prototype instrument we were using.
The detergent-sensitive enzyme-inhibition assay, although it is admittedly less direct than
physical measurement of particle formation, was found to be more robust, faster and more
amenable to miniaturization. This protocol was previously described in ref. 5, and general
discussions of the approach are presented in refs. 7 and 10. Note that in ref. 5, the concentration
of detergent used was 0.1% vol/vol Triton X-100. Here we recommend a lower percentage,
0.01% vol/vol, which is better tolerated by the enzyme.
Experimental design
The counter-screen relies on the detergent-sensitive nature of aggre-gate-based inhibition:
molecules that inhibit an enzyme in the absence, but not the presence, of detergent are likely
to be inhibiting via the formation of promiscuous aggregates. Thus, the first step of the
experiment is to screen molecules for inhibition. AmpC β-lactamase (AmpC) is a convenient
choice of enzyme because it has been extensively studied for aggregate-based inhibition, and
because it is one of the more sensitive enzymes tested for this effect. In principle, however,
any soluble enzyme can be used for this protocol, as compound aggregation is a physical
property of small molecules in aqueous buffers. The only limitation to using other enzymes is
how well tolerated Triton X-100 is in different assay systems. For systems that do not tolerate
non-ionic detergent, 1 mg ml-1 BSA might be considered as a replacement. However, BSA can
sequester monomeric small molecules, so this should be used with caution.
The experiments described in this protocol are also amenable to either scale-up or scale-down.
There are no problems running these experiments in lower-throughput cuvette-based formats.
Higher-throughput formats, such as 1,536-well plates, are conceivable. We recommend that
experiments be conducted at least in duplicate, to ensure statistical significance — aggregating
molecules can be capricious, and are sensitive to assay conditions and target enzyme
concentration, as well as centrifugation, vortexing and other invasive mixing procedures.
If this protocol is applied to a different enzyme system or significantly different assay
conditions, it might be necessary to derive new cut-offs for detecting aggregate-based
inhibition. To establish what constituted significant inhibition in the development of our
technique, we used a panel of known aggregators and non-aggregators. These molecules are
listed on our website (http://shoichetlab.compbio.ucsf.edu) and can be screened as positive
controls to identify the window of relevant inhibition (see ANTICIPATED RESULTS and ref.
5).
Finally, screening for inhibition in the presence of detergent can be carried out independently
from screening for inhibition in the absence of detergent — each experiment can be done
separately and the procedure for each is the same. However, when adding detergent
individually to each well, add it to buffer before any other components.
MATERIALS
REAGENTS
•Reaction buffer: 50 mM potassium phosphate (25 mM KH 2PO4 + 25 mM
K2HPO 4), pH 7
•Detergent reaction buffer: 50 mM potassium phosphate + 0.01% Triton X-100 vol/
vol, pH 7 ▲ CRITICAL Aqueous Triton X-100 solutions become less effective over
time and should be made fresh dailyFeng and Shoichet Page 2
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•Protein: 0.00162 mg ml-1 AmpC, containing 0.0006% Triton X-100 vol/vol in KPi;
0.00162 mg ml-1 for 30×; store at 4 °C; the preparation of AmpC has been previously
described6,11; exploratory amounts are available from the Shoichet Laboratory ▲
CRITICAL Prepare working enzyme stocks daily from higher concentration stocks
(>1 mg ml-1). Enzyme will gradually adsorb to the surface of the container, thus
decreasing the concentration of free enzyme. The small amount of detergent added
to the enzyme stock will attenuate this effect
•Substrate: nitrocefin; 5 mM stock in DMSO; Remel/Oxoid (cat. nos. 651063/
BR0063A, respectively) ▲ CRITICAL Store solid stocks at 4 °C and DMSO stocks
at -20°C
•Compounds: 10 mM stocks in DMSO ▲ CRITICAL DMSO concentration in the
reaction should be minimized. AmpC tolerates concentrations <4% vol/vol without
serious effect
EQUIPMENT
•A UV-visible plate reader
•A 96-well-format liquid-handling instrument, such as the Biomek FX (Beckman)
•96-well-format tips for use with a liquid-handling robot (e.g., Molecular Bioproducts
cat. nos. 919-262-05 and 918-262-05)
•96-well plates for reagents and dilutions (e.g., Grenier 96-well plates, cat. no. 65020)
•UV-transparent 96-well plates for reactions (e.g., Corning cat. no. 3679)
PROCEDURE
A detergent-based counter-screen for aggregation-based inhibition
1.Add buffer to each well. For a final reaction volume of 150 μl, pipette 142 μl to ×
μl, where × is the amount of compound to be added.
2.Add 5 μl of 30× enzyme solution to the wells.
3.Add × μl of compound (or DMSO for uninhibited positive control) to the wells.
▲ CRITICAL STEP Only a few controls are necessary to establish the uninhibited
rate of the reaction.
4.Mix by pipetting up and down.
5.Incubate compounds and enzyme for 5 min.
▲ CRITICAL STEP Aggregate-based inhibition is time dependent; therefore, the
incubation time is key.
6.Add 3 μl of 5 mM nitrocefin.
7.Mix by pipetting up and down.
8.Monitor reaction at 482 nm for five minutes. This is the emission maximum for
nitrocefin—however, off-peak wavelengths can also be used.
• TIMING
Timeline:
Steps 1-4: 5 minFeng and Shoichet Page 3
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Step 5: 5 min
Step 8: 5 min
? TROUBLESHOOTING
See Table 1.
ANTICIPATED RESULTS
Inhibition
The initial rate of each reaction is the slope of the best-fit line to the early kinetic data. The
percentage of inhibited enzyme is defined as:
% Inhibition = 100∗(1 −vi
vc)
Here, vi and vc are the inhibited and uninhibited rates of reaction, respectively. We defined
statistically significant inhibition as greater than 23.8%, based on the behavior of several known
aggregators and non-aggregators at 30 μM (refs. 5,6). Less than 11% inhibition was considered
insignificant. Intervening amounts of inhibition were ambiguous, and could indicate a tendency
to aggregate at higher concentrations. These cut-offs were statistically obtained from the
behavior of compounds in our hands and on our instruments, and thus only represent guidelines.
These cut-offs might not be extendable to other enzyme systems, or to significantly different
assay conditions or concentrations.
Effect of detergent
We consider a broad range of decrease in inhibition upon the application of detergent to be
significant. Most aggregators show a greater than twofold decrease in percentage inhibition
upon the application of 0.01% Triton X-100, vol/vol although not all aggregators have the same
sensitivity to detergent5,7.
If a molecule exhibits significant inhibition of AmpC, which is diminished by detergent, it is
almost certainly acting as an aggregation-based inhibitor. Marginal detergent sensitivity should
not be considered a confirmation of true inhibition — some aggregators, such as Congo red,
require 0.1% Triton X-100 vol/vol before inhibition is fully reversed. Addressing this widely
varying sensitivity definitively might require multiple experiments with different
concentrations of detergent or compound. That said, aggregation at one concentration does not
rule out a specific mechanism at a lower concentration. If further experimental confirmation
of aggregation is required, other hallmarks of this phenomenon can easily be assayed — e.g.,
the presence of particles detectable by light scattering, the time dependence of inhibition or
the dependence of inhibition on enzyme concentration (see below). However, the detergent-
dependent enzyme assay is relatively definitive and is the easiest to incorporate into a large-
scale screening campaign. It represents a fast and simple way to screen for promiscuous
aggregate-based inhibition.
Follow-up experiments for confirmation of aggregate-based inhibition
When testing only a small number of interesting molecules for aggregation-based inhibition,
more detailed (and time-consuming) investigation might be called for. The following
experiments, in decreasing order of facility, might be useful.
1.Is inhibition significantly attenuated by small amounts of non-ionic detergent? If so,
the compound is likely to be acting through aggregation. We typically use 0.01%Feng and Shoichet Page 4
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Triton-X 100 vol/vol, while others favor Tween-20 or CHAPS12, and we have also
used saponin10 and digitonin. Other non-ionic detergents might also work.
2.In assays that cannot tolerate detergent (e.g., cell-based assays), it might be possible
to use high concentrations of serum albumin6. This is currently under investigation
— a drawback of this method is that albumin can also sequester well-behaved
molecules.
3.Is inhibition significantly attenuated by increasing enzyme concentration? If so, the
compound is likely to be an aggregator. Except when the receptor concentration-to-
Ki ratio is high13, increasing receptor concentration should not affect percentage
inhibition. Of course, when the receptor is membrane-bound or intracellular, this is
difficult to probe.
4.Is inhibition competitive? If so, the compound is unlikely to be an aggregator. Does
the inhibitor retain activity after spinning for several minutes in a microfuge? If not,
particle formation is likely (see below).
5.Can you directly observe particles in the 50-1,000 nm size range? We have typically
used DLS for this. Formation of particles does not guarantee promiscuous inhibition,
but it is a worrying sign.
6.Is the dose-response curve unusually steep? There are classical reasons for steep dose-
response curves13, but it too is a worrying sign.
Few, if any, of these experiments are definitive by themselves, although the detergent test is
fairly reliable. When several of these tests are combined, they are strong indicators of
aggregation-based or non-aggregation-based mechanisms of action. Further discussion of
characteristic features of aggregation-based inhibition can be found in refs. 6-9.
Acknowledgements
ACKNOWLEDGMENTS We thank A. McReynolds and K. Coan for reading this manuscript, K.C. for thoughtful
discussions and GM71630.
References
1. Hajduk PJ, Huth JR, Fesik SW. Druggability indices for protein targets derived from NMR-based
screening data. J. Med. Chem 2005;48:2518–2525. [PubMed: 15801841]
2. Rishton GM. Reactive compounds and in vitro false positives in HTS. Drug Discov. Today 1997;2:382–
384.
3. Walters WP, Namchuk M. Designing screens: how to make your hits a hit. Nat. Rev. Drug Discov
2003;2:259–266. [PubMed: 12669025]
4. Feng B, Shoichet BK. Synergy and antagonism of promiscuous inhibition in multiple-compound
mixtures. J. Med. Chem 2006;49:2151–2154. [PubMed: 16570910]
5. Feng BY, et al. High-throughput assays for promiscuous inhibitors. Nat. Chem. Biol 2005;1:146–148.
[PubMed: 16408018]
6. McGovern SL, Caselli E, Grigorieff N, Shoichet BK. A common mechanism underlying promiscuous
inhibitors from virtual and high-throughput screening. J. Med. Chem 2002;45:1712–1722. [PubMed:
11931626]
7. McGovern SL, Helfand BT, Feng BY, Shoichet BK. A specific mechanism of nonspecific inhibition.
J. Med. Chem 2003;46:4265–4272. [PubMed: 13678405]
8. McGovern SL, Shoichet BK. Kinase inhibitors: not just for kinases anymore. J. Med. Chem
2003;46:1478–1483. [PubMed: 12672248]
9. Seidler J, McGovern SL, Doman T, Shoichet BK. Identification and prediction of promiscuous
aggregating inhibitors among known drugs. J. Med. Chem 2003;46:4477–4486. [PubMed: 14521410]Feng and Shoichet Page 5
Nat Protoc . Author manuscript; available in PMC 2006 December 27.
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10. Seidler J, McGovern SL, Doman TN, Shoichet BK. Identification and prediction of promiscuous
drugs. J. Med. Chem 2003;46:4477–4486. [PubMed: 14521410]
11. Weston GS, Blazquez J, Baquero F, Shoichet BK. Structure-based enhancement of boronic acid-
based inhibitors of AmpC β-lactamase. J. Med. Chem 1998;41:4577–4586. [PubMed: 9804697]
12. Ryan AJ, Gray NM, Lowe PN, Chung C. Effect of detergent on “promiscuous” inhibitors. J. Med.
Chem 2003;46:3448–3451. [PubMed: 12877581]
13. Straus OH, Goldstein A. Zone behavior of enzymes: illustrated by the effect of dissociation constant
and dilution on the system cholinesterase-physostigmine. J. Gen. Physiol 1943;26:559–585.Feng and Shoichet Page 6
Nat Protoc . Author manuscript; available in PMC 2006 December 27.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript | e88e9e2ebb16c1087f8fa965fe46da1450ba2498 | page_0005 |
Figure 1.
Transmission electron micrograph of aggregates of tetraiodophenolphthalein (dark edged
circles) associated with β-galactosidase. This association inhibits the enzyme by sequestering
it from substrate, although whether these are adsorption or absorption effects remains uncertain
at this time. Bar, 200 nm. Reproduced from ref. 7Feng and Shoichet Page 7
Nat Protoc . Author manuscript; available in PMC 2006 December 27.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript | e88e9e2ebb16c1087f8fa965fe46da1450ba2498 | page_0006 |
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFeng and Shoichet Page 8
TABLE 1
Troubleshooting table.
PROBLEM POSSIBLE REASON SOLUTION
Reaction rate is low or drops over the
course of multiple experimentsEnzyme adsorption to the reagent container Prepare new enzyme stocks throughout the
experiments Reuse the enzyme reagent container
High variation in control reaction rates Poor mixing Mix more thoroughly
Poor reversibility in detergent
experimentsTriton X-100 has gone bad Prepare fresh Triton X-100
Sudden large jumps in absorbance at 482
nmPopping bubbles from the presence of Triton
X-100Mix less vigorously and ensure mixing takes place
below the liquid level of the well
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ORiK:ASSESSMENT OFCARDIAC MURMURS 7
*observed, letmeremindyouthatinourstudies
uponcatalytic elements-manganese andzinc
-inthecultivation ofAspergillus Nigerwe
,clearly distinguished, M.G.Bertrand andI,
thatinterdependance oftheconstituents ofthe
nutritive medium. Itisnotenoughtogivethe
plantallthatitrequires; theelements ofthe
medium mustalsobepresent inappropriate
.quantitative ratios.Idonotmeantosaythat
theplantwillnotliveiftheseratiosarenot
respected (theyarenotobserved innature),
butthattheoptimum growthofplants,and
-themosteconomical utilization offood,will
correspond togivenratiosbetween allthe
.elements, theplasticaswellasthecatalytic.
Forzinc,Ihaveclearlystatedthatitinter-
-feres,directly orindirectly, intheutilization
*ofsugar.
Thatideaofthenecessity ofanalimentary
*equilibrium foranimals andman(whois
indeedtheanimalthatfeedshimself inthe
leastrationalmanner) hasalsooccurred toour
mindinaratherconfused andindefinite way.
'Thereasonisthattheproblem isheremore
complicated andexperiment moredifficult.
:Still,thethought isbecoming gradually moreprecise. Thezootechnicians arewellaware
thatthereisacertainoptimum ratiobetween
theproteicfoodandthenon-proteic food,be-
tweentheintakeofphosphorus andthatof
calcium. Mme.Randoin andM.Simonnet have
recently givenaremarkable instance ofthe
necessity ofsuchanequilibrium. Havingfound
thatthevitaminB,is,amongotherthings,the
factorforutilization ofsugar,theystatedthat
acertainquantitative ratiobetween factorB
andtheglucide ofthedietmustexist.
Equilibrium ofcellular composition, equilib-
riumofcomposition ofthefood,and,between
thetwo,ifImaysayso,equilibrium ofcom-
position oftheinteriormedium, herearethe
threenecessities, answering andco-ordinating
oneanother.
Itseemstomethatanotionlikethisis
worthcallingtotheattention ofbiochemists
anidphysiologists, anditisbecause Iamdeeply
imbuedwiththatthought, thatIhaveallowed
myselftogosofarinspeaking toyouaboutit.
Thishasbeenformeanopportunity toap-
preciate, together withthecordiality ofyour
welcome, thekindattention youhavefavoured
mewith.Letmethankyouheartily forit.
THEASSESSMENT OFCARDIAC MURMURS
BYJAMESORR,M.B.,M.R.C.P. (EDIN.),
Physician, JamesMackenzie Institute forClinical Research,
St.Andrews, Scotland
'TIHEsignificance ofacardiacmurmur ises-
sentially diagnostic. Fromthepointofview
*ofprognosis, themurmur perseisoflittlevalue.
Ineverycardiaccondition, inwhichamurmur
ispresent, prognosis depends upontherecog-
-nitionandproperappraisement ofsuchsymp-
tomsasindicate thedegreeofefficiency ofthe
ventricular muscle. Toelicitthisinformation
-thepatient mustberegarded asawhole. It
isimpossible tobaseacorrectprognosis onthe
-presence orabsence ofanyonesignorsymp-
-tom.Further, thesymptoms uponwhichdiag-
nosisisbasedarenotnecessarily thoseupon
whichprognosis isassessed, and,inthecourse
'ofclinicalexamination, itisdesirable tosepar-
'ateclearlythesetwogroupsofsymptoms.
Diagnosis is,inthemain,baseduponthe
-recognition ofthestructural changetowhichanorganhasbeensubjected. Prognosis, onthe
otherhand,involves anappreciation ofthe
extenttowhichthefunctional efficiency ofthe
organhasbeenimpaired. Diagnosis depends
largelyuponthepresence orabsence ofcertain
physical signs;prognosis, uponthesubjective
symptoms ofwhichthepatientmakescomplaint.
Physical findings arereferable directly tothe
organinvolved, whereas thesymptoms offunc-
tionalembarrassment are,intheearlierstages
atleast,manifested byorgansotherthanthat
whichistheseatofthepathological change.
Thusdiagnosis, however correctitmaybe,does
notcarryitsprognosis withit.Mass-prognosis
iseasilyarrivedatonthebasisofpathological
diagnosis. Itispossible fromactuarial figures
toarriveatanexactestimate ofthemortality
andmorbidity incidence ofanvgivencondition, | f6678406e03c649487c40176ec9c776773a09abf | page_0000 |
THECANADIAN MIEDICAL ASSOCIATION JOURNAL
butphysical signsgivelittlehelpinframing
theindividual prognosis whichthepatientseeks.
Thiscanonlybeobtained byacarefulestimate
ofthefunctional efficiency oftheorgancon-
cerned. Inthecaseoftheheartthisresolves
itselfintoanestimation oftheefficiency ofthe
ventricular muscle,andmanymeanshavebeen
devised tothisend.Someofthesearebased
uponthereaction oftheheart,oroftheblood
pressure tomuscular exertion; othersuponcer-
tainabnormalities demonstrable intheelectro-
cardiogram. Noneofthesearealtogether re-
liable,andtheirfindings arefrequently belied
bythesubsequent historyofthepatient. The
onlytestuponwhichreliance canbeplacedis
theclinical testoftheresponse ofthepatient
toordinary muscular exertion, i.e.,themanner
inwhichheisabletoperform hisusualdaily
activities asindicated bythesymptoms and
sensations towhichtheirperformance gives
rise.Apatientwhosemyocardium isbecoming
impaired willrecognize alimitation ofadefinite
kindinhiscapacity foreffortlongbeforeex-
amination willrevealanything amiss. This
method hasthefurtheradvantage thatitis
individual, thepatientbeingcompared withhis
ownprevious normal. Theso-called "exercise
tolerance" testshavethedisadvantage thatthey
lacktheessential factorofcontrol. Thepatient
istestedagainst astandard towhichhisrela-'
tioninthedaysofhisnormalhealthisunknown.
Noreaction whichapatientshowsinresponse
tomuscular exertion caneverberegarded asun-
important, buttherealquestion is,howthe
reaction atthetimeofexamination compares
withasimilarreaction inhealth. Inthecase
oftheclinicaltest,thepatient cansupplythis
information withgreataccuracy.
Muchingenuity hasbeenexercised inthe
classificationi ofmurmurs, especially systolic
murmurs, andvarious conclusions havebeei
basedupontheseclassifications. Thus,systolic
murmurs havebeendivided intothosewhich
followthefirstsound,thosewhichreplace it,
andthosewhichoccurconcurrently withit.
Again,theyhavebeenclassified asblowing,
roughormusical, according totheirquality.
Whatever descriptive ordiagnostic valuesuch
classification maypossess, itisentirely in-
adequate asameansofprognostic assessment.
Theonlyfactorwhichisofanyimportance for
thispurpose isthecapacity oftheventricularmuscle tomaintain anadequate circulation.
Noconsistent relation between thiscapacity
andanyofthevariations whichthemurmur
mayshowhaseverbeendemonstrated.
Mackenzie classified cardiac murmurs into
threegroups-the physiological, thefunctional,
andtheorganic. Thisclassification wasintro-
ducedasareaction against thethencurrent
viewthatallmurmurs werenecessarily of
seriousimport.
Thephysiological murmuir occursincon-
ditionsofperfect health,inwhichtheheart
responds normally toeverydemand madeupon
it.Iffromaccident orothercausedeathoccurs,
theheartshowsnoabnormality onpostmortem
examination. Postural systolic murmurs and
respiratory murmurs belongtothisgroup.
Functional murmurs arenotassociated with
anyorganic changeinthevalve.Theyusually
occurinassociation withadegreeofdilatation
oftheheart,andarepresumably duetorelaxa-
tionofthemiitralring.Theyareaccompanied
withsomelimitation oftheresponse toeffort.
Physiological andfunctional murmurs areal-
wayssystolic intime.Functional diastolic
murmurs havebeendescribed, buttheiroccur-
renceissorareastobenegligible.
Organic murmurs areassociated withsecond-
arychanges inthevalve,generally ofafibrotic
ordegenerative kind.Thedistinction betweeni
afunctional andanorganic murmur isnot
alwayspossible. Indoubtful cases,thehistory
isofthehighest importance. Aroughor
musical qualityisperhaps morecommon in
organic murmurs. Theimportance ofan
organic murmur isthatitindicates thatthe
valveis,orhasbeen,theseatofdisease, and
raisesthequestion astowhether otherand
morevitalpartsoftheheartmaynothave
beeninvaded also.Toassessthevalueofany
murmur, itmustbeconsidered fromtwopoints
ofview,viz.,thecondition oftheorifice,andthe
condition ofthemyocardium.
APPLICATION
Diastolic Murrnurs.-Diastolic murmurs are
nearlyalwaysorganic inorigin,andindicate
lesions ofvalveswhichenmbarrass theheart
andtendtotheproduction ofheartfailure.
Amurmur occurring during diastole means
thatoneoftwoconditions ispresent, either
mitralstenosis oraorticregurgitation.
Murmurs ofmitralstenosis. Theearliest8 | f6678406e03c649487c40176ec9c776773a09abf | page_0001 |
ORR:ASSESSMENT OFCARDIAC MURMURS
signofmitralstenosis istheoccurrence ofa
shortmurmur immediately preceding thefirst
sound. Atfirstthismurmur isvariable, but
soonbecomes persistent. Occasionally, thefirst
indication ofthecondition istobefoundina
reduplication ofthesecondsound. Asthe
stenosis advances ashortdiastolic murmur is
added,whichgradually increases inlengthas
thecontraction ofthevalveorificeproceeds,
tntilitoccupies thewholeofthediastolic
pause.Shouldfibrillation oftheauriclesuper-
vene,thepresystolic element ofthemurmur
disappears, leavingonlythediastolic murmur.
Incaseswheretheonsetofthepresystolic
murmur hasbeenobserved afairideaofthe
progress ofthecondition canbeobtained by
observing therateatwhichthesechanges
occur. Ifthecaseisseenatalaterstage,a
history canoftenbeobtained ofanattackof
rheumatic feverwhichpresumably setupthe
valvemischief, andtherateofprogress
assessed inasimilar manner. Thestenosis
beingduetocicatrical contraction, thepre-
systolic murmur isneverheardduring the
initialillness,butfollowsitafteraconsiderable
interval oftime.Exactevidence oftherate
atwhichthemurmurs ofmitralstenosisdevelop
isscanty,butfromasmallseriesofcasesin
whichthiscondition hasbeenobserved tofollow
rheumatic fever,apbriodoftwotofiveyears
elapsedbetween theattackandtheappearance
ofthepresystolic murmur.
Inonecaseofrheumatic endocarditis, in
whichtheheartwasexamnined atfourday
intervals overaperiodofnearlytwoyears,
thefirstappearance ofthemurmur ofendo-
carditis wasnotedontheninthdayofthe
illness(January 12,1922). Anaorticre-
gurgitant murmur wasnotedonFebruary 7.
1923.Areduplication ofthesecondsoundat
theapexsuggestive ofmitralstenosisappeared
July27,1923.Deathoccurred fromerysipelas
andsepticpneumonia November 1,1923,no
presvstolic murmur havingdeveloped upto
thattime.Postmortem examination showed
thetypical appearance ofold-standing endo-
carditis, narrowing ofthemitralorifice, the
cuspsofthevalvebeingthickened, fibrous,
shrunken, andfusedwithoneanother atthe
adjacent margins. Theaorticvalvewasin-
competent, thecuspsbeingthickened attheir
margins andslightly shrivelled.Thepresence ofapresystolic murmur isin
itselfausefulstarting pointforassessing the.
progress ofthecondition. Thus,mitralstenosis
inyoungpeople, accompanied bypresystolic
anddiastolic murmurs, isaverygravecondi-
tion;whereas inamiddle-aged patient, with
ahistoryofrheumatic feverinchildhood, and
onlyapresystolic murmur present, therateof
stenosis isobviously slowandmayevenbe
stationary. Incaseswhichareprogressive
thereareinvariably factorspresent otherthan
thevalvechange. Rheumatic infection affects
allthetissuesoftheheartandusuallyproduces
adegree ofimpairment oftheventricular
muscle. Itisuponthisfactorthattheprog-
nosisinthemaindepends. Evidence of
affection oftheconducting system-or genetic
system,asMackenzie morecorrectly calledit,-
istobefoundinthepresence ofthemid-
diastolic murmur. Thismurmur isseparated
fromboththefirstandsecondsounds. Mac-
kenziedemonstrated thatitcoincided intime
withtheauricular systole, itsoccurrence in
mid-diastole beingduetothefactthatthe
systoleoftheauriclewasseparated byapause
fromthatoftheventricle. Thepresence ofa
mid-diastolic murmur, therefore, isevidence
notonlyofthepresence ofmitralstenosis, but
alsoofadegreeofheart-block. Therearethus
threefactorsofimportance intheassessment
ofthemurmurs ofmitralstenosis: (1)the
natureofthemurmur present, asindicating the
condition oftheorifice; (2)therapidity with
whichthesequence ofmurmurs isdeveloping;
and(3)thecapacity oftheheartmuscleto
carryonanefficient circulation, inspiteofthe
obstruction causedbythevalvular defect. If
fibrillation hasoccurred, anadditional em-
barrassment isthrown ontheventricular
muscle, andintheseconditions theprognosis
mustbebasedonthethirdfactor,andupona
consideration oftheresponse totreatment.
Patients withmitrallesionsrarelydieintheir
firstattackofheart-failure, however severeit
maybe,andnoadequate prognosis insuch
casesispossible untiltheresponse totreatment
hasbeencarefully observed. Aconsiderable
proportion efthesubjects ofmitralstenosis do
notdiefromheart-failure atall-according to
Cabotaboutone-half-but fromsomeintercur-
rentaffection, orfrommalignant endocarditis.9 | f6678406e03c649487c40176ec9c776773a09abf | page_0002 |
THECANADIAN MEADICAL ASSOCIATION JOURNAL
Murmurs ofaorticr.egurgitation. Aortic
regurgitation is,asarule,averyserious con-
dition,thoughthisisnotinvariably thecase.
Manyelderlypeopleshowing thecharacter-
isticmurmurs ofaorticregurgitation lead
healthy andevenstrenuous lives,whileothers
showing identical murmurs passrapidly into
acondition ofextreme heart-failure. No
validprognosis canthusbebaseduponthe
merepresence ofaregurgitant murmur. Nor
doestheamount ofregurgitation appear tobe
afactorofanyparticular significance. Even
ifitwere,thepossibility ofgaugiilg theextent
oftheregurgitation clinically isanexceedingly
doubtful one.Theloudness ofthediastolic
murmur, itslength,thepresence orabsence
ofthesecondsound,havebeensuggested as
possible indications oftheextentofthere-
gurgitation. Thedifference between thesys-
tolicanddiastolic arterial pressure, i.e.,the
pulsepressure, hasbeenutilized forthesame
purpose. Themaindifficulty withsuchindica-
tionsisthattheyarenotalwaysconsistent in
thesamepatient. Thus,alongdiastolic mur-
mur,suggestive ofalargeleak,maybeasso-
ciatedwithamoderate pulse-pressure. Apart
fromthefactthatthereisnloreliable method
bywhichtheamount ofregurgitation canbe
estimated clinically, post-mortem evidence gives
nosupport totheviewthattheextentofthe
damage tothevalvenecessarily corresponds
withthedegreeofheart-failure observed during
life.
Thecondition ofthepulse,though oflittle
helpasanindexofthedegreeofregurgitation,
is,nevertheless, afactorofconsiderable prog-
nosticimportance. Asarule,whenthe
systolicpressure isveryhighandthediastolic
pressure verylow(190-60), otherevidence of
heart-failure ispresent. Amoderate difference
between thesystolic anddiastolic pressures
(180-90) isquiteconsistent withanefficient
heart,while,insomecases,thedifference is
equaltothenormal. Intheselastinstances
thepulsehaslittleofacollapsing character,
though, asVaquezpointsout,theabruptrise
ofpressure witheachpulsebeatisaptto
conveytothefingeranimpression ofacollaps-
ingquality. Whenaorticregurgitation is
arterial inorigin,thediastolic pressure maybe
consistently high.Thepulsecondition, there-
fore,mustbeconsidered inconjunction withtheothersymptoms present. Among these
othersymptoms thesizeoftheheartisan
important guide. Inthecaseswholead
vigorous lives,freefromanysuggestion of
heartfailure,theleftventricle isnevermore
thanslightlyhypertrophied, themurmurs being
dependent uponchanges whichare,inthe
main,limited tothevalves. Inmoreserious
conditions, alldegrees ofhypertrophy maybe
present.
Themostimportant prognostic factorinthis,
asinallothercardiaeconditions, isthestate
oftheventricular muscle, asshownbythe
response ofthepatient toordinary effort.
Contrary towhatobtainsinmitraldisease,the
heartfailure ofaorticregurgitation isless
susceptible totreatment, andrecovery isless
frequent andlesspronounced thaninheart
failureresulting fromanyothercondition.
Evenwhenheartfailureisextreme inmitral
stenosis withauricular fibrillation, agoodre-
coverymayoftenoccur,butinaorticdisease
withauricular fibrillation theheartfailureis
usuallysteadily progressive, anditisseldom
possible tocheckitsprogress.
SystolicMurmurs.-The assessment ofsystolic
murmurs isconducted onentirely thesame
principle ashasbeenindicated indealingwith
diastolic murmurs. Systolic murmurs, however,
present certaindifficulties.of theirown,owing
largely tothefrequency withwhichtheyare
functional orphysiological inorigin. Inthe
caseofasystolic murmur arisinginthecourse
ofanacuteaffection, thedistinction between a
functional andorganic murmur maybevery
difficult, and,particularly inthecaseofrheu-
maticfever,thegravestissuesmaydependupon
itsproperinterpretation.
Aort`cstenosis. Inthecaseofasystolic
murmur discovered onordinary examination,
theimportant pointtodefineiswhether ornot
itisassociated withsignsindicating thatthe
ventricle isbeinghampered initswork.The
murmur ofaorticstenosis isacaseinpoint.A
systolic murmur atthebaseoftheheartisin
themajority ofinstances functional orphysio-
logicalinorigin,andisnotassociated withany
evidence ofinefficiency oftheventricular muscle.
Iftheaorticorificeisnarrowed, theresponse to
effortwillbelimitedandtheleftventricle en-
larged. Apulsetracingmayshowtheanacrotic
pulsesometimes associated withthiscondition.10 | f6678406e03c649487c40176ec9c776773a09abf | page_0003 |
ORR:ASSESSMENT OFCARDIAC MURMURS
Aorticstenosisoccurring byitselfisanexceed-
inglyrarecondition.
Mitralincompetence. Mitralincompetence
byitselfprobably neverconstitutes aserious
embarrassment totheheart.Therecentwork
ofCabotwouldappeartoindicate thatmitral
regurgitation istherarestofallvalvular
affections, andindeedmaybesaidhardlyto
existatallasapathological entity. Howfar
itexistsasaclinicalentityisperhaps another
proposition, fortheredoesnotseemtobeany
certainmeansofestimating afterdeathwhat
thecompetence ofthevalvemayhavebeen
duringlife.Whilepathological evidence of
organic mitralinsufficiency maybescanty,
functional mitralregurgitation duetorelaxa-
tionofthemitralringmaybeofmorefre-
quentoccurrence. Thequestion is,however, of
littlepractical importance, forinestimating
thesignificance ofsystolic murmurs atthe
mitralvalveitmakeslittledifference whether
ornotregurgitation ispresent, provided that
theheartmuscleshowsnosignsofinefficiency.
Asystolic murmur discovered accidentally in
anindividual whoisotherwise inperfect
health,andwhoshowsnolimitation inhis
capacity foreffort,maybecompletely dis-
regarded. Ifitoccursinassociation with
signsindicating alimitation ofresponse to
muscular effort,especially ifthelimitation be
thatofbreathlessness orpaininthechest,the
prognosis shouldbebaseduponthesesigns,and,
-notonthemurmur, thepresence orabsence of
whichdoesnotaffecttheissuetoanydegree.
Systolic murmurs arising inthecourseof
aeuteillness. Inthecourseofacuteillnessa
systolic murmur ispronetodevelop, especially
inyoungsubjects, andisfrequently associated
withadegreeofdilatation oftheheart.The
murmur inthemajority ofthesecasesisfunc-
tionalinorigin,andusuallydisappears when
convalescence isestablished. Afunctional
murmur arising insuchcircumstances isun-
accompanied byanysignsofdeterioration in
thepatient's generalcondition. Thecourseof
theillnessisunaffected, therateofthepulse
remains unaltered, andthepatient progresses
normally towards convalescence. Coombslays
stressonthefactthattheinorganic murmur is
audible inallareasandthattheheartisnot
asaruleenlarged. Ifamurmur iscausedby
alesionwhichembarrasses theheartinitswork,thechamber affected willalterinform,
eitherbydilating orbybecoming hyper-
trophied. Theabsence, then,ofanyalteration
inthesizeoftheheartisevidence thatthereis
littleembarrassment. Thepresence ofthe
general characteristics ofinorganic murmurs,
suchaspostural variation, etc.,areimportant
confirmatory signs. Thedistinction between
functional andendocardial murmurs isoften
impossible duringtheacutephaseofanillness.
Itisnecessary asaruletosuspend judgment
untilthesubsidence oftheacuteattack,when
thepointsofdistinction become moreevident.
Thisisparticularly sointhecaseofrheumatic
fever. Inthiscondition, asinotheracute
affections, afunctional systolic murmur is
frequently present. Ontheotherhand,the
liability ofthehearttorheumatic infection
suggests apossible endocardial originforany
murmur whichmaydevelop. Itisobviously of
thehighest importance thatthesetwocondi-
tionsshouldbeclearlydifferentiated. Thereis
noonecriterion whichisapplicable toallcases.
Eachmustbejudged onitsownmerits,but
therearecertaingeneral considerations which
helptowards acorrectdecision.
Themodeofonsetofthemurmur, andthe
conditions associated withitsdevelopment,
provide themostreliable indications forthis
purpose. Themurmur ofendocarditis usually
beginswithablurring ofthefirstsoundinthe
mitralarea,andisassociated withpersistent
rapidity ofthepulseandadecline, oratleast
awantofimprovement, inthecondition ofthe
patient. Theblurred firstsoundsoongives
placetoadefinite murmur andisassociated,
asarule,withperceptible dilatation ofthe
heart.Dilatation oftheheartmayoccurin
conjunction withamurmur whichispurely
functional, butthesetwosigns,pluspersistent
rapidity ofthepulseandwantofimprovement
inthegeneralcondition, arehighlysuggestive
oftheonsetofendocarditis. Shouldrheumatic
nodules bepresent, thediagnosis ispractically
certain. Endocarditis, whichismoreproneto
occurinyoungsubjects, generally makesits
appearance duringthefirstfortnight ofrheu-
maticfever. Themurmur itselfmaynot
appearforaconsiderable time,andtheheart
shouldbecarefully watched foritsoccurrence
untilwellafterthejointsymptoms have
subsided.11 | f6678406e03c649487c40176ec9c776773a09abf | page_0004 |
© 2008 Emery et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
which permits unrestricted noncommercial use, provided the original work is properly cited.Vascular Health and Risk Management 2008:4(1) 259–262 259CASE REPORT
A hazardous fi nding of a rare anomalous left main
coronary artery in a patient with a secundum atrial septal defect
Michael Emery1
Waqas Ghumman1
Shawn T eague2
Jo Mahenthiran1
1Krannert Institute of Cardiology,
2Department of Radiology, Indiana
University School of Medicine, Indianapolis, Indiana, USA
Correspondence: Jo Mahenthiran
Indiana University School of Medicine, Krannert Institute of Cardiology, 1801 N Senate Blvd, MPC2, Suite 4000, Indianapolis, IN 46202, USAT el +1 317 962 0539Fax +1 317 962 0116Email [email protected]: A 23-year-old male referred for evaluation of a “choking” sensation with exertion and
a murmur. A transthoracic echocardiogram demonstrated right atrial and ventricular dilatation, right ventricular volume overload, and a large secundum atrial septal defect (ASD) with left to right shunt and a calculated pulmonary-to-systemic blood fl ow ratio ( Qp/Qs ) estimated at 2.3 to
1. Cardiac catheterization also demonstrated evidence of the ASD with Qp/Qs of 4.6 to 1 with a
signifi cant step-up in oxygen saturation at the right atrial level. Additionally, an anomalous left
main coronary artery (ALMCA) origin from the anterior right coronary cusp was suspected. Using 64-slice multidetector computed tomography coronary angiography (CCTA) the left main coronary artery was seen to arise from the right coronary cusp then traverse between the pulmo-nary trunk and the proximal ascending aorta before bifurcating into the left anterior descending and circumfl ex arteries that followed their normal courses distally. Based on the high risk nature
of associated sudden death from an anomalous left main coronary artery (ALMCA) coursing between the aorta and the pulmonary trunk, the patient underwent surgical re-implantation of the ALMCA to the left coronary cusp and repair of the ASD. This case highlights a rare fi nding of
a hazardous ALMCA in a patient with a secundum ASD and the utility of CCTA in evaluating the course of coronary anomalies along with other cardiac pathology.Keywords: atrial septal defect, anomalous coronary artery, congenital heart disease
We report a case of a 23-year-old Hispanic male who was referred for evaluation
of a “choking” sensation with exertion and a murmur. The patient’s past medical history was signifi cant for hypertriglyceridemia for which he was on gemfi brozil.
He described a sensation of choking with mild dyspnea during activities that would promptly resolve with rest. He denied any other associated symptoms such as chest discomfort, diaphoresis, syncope, lightheadedness, palpitations or nausea. These symptoms had been present for several months and led him to stop participating in strenuous activities.
On physical exam, pulse was 72 beats per minute and blood pressure
134/64 mmHg. His apical impulse was not clearly palpable, but there was a prominent right ventricular lift. Cardiac auscultation was notable for regular rate and rhythm and a narrowly split second heart sound with reduced respiratory variation. An II/VI early systolic ejection murmur; a soft, early decrescendo diastolic murmur along the left sternal border; and a prominent mid-diastolic murmur at the left lower sternal border were audible. Lungs were clear and the rest of his exam was unremarkable. 12-lead electrocardiogram showed normal sinus rhythm with Wenckebach type atrio-ventricular block, incomplete right bundle branch block and non-specifi c ST-T
wave changes. | 12439b7452d8cc28f773b2e3fbe7a31ebaebff94 | page_0000 |
Vascular Health and Risk Management 2008:4(1) 260Emery et al
Figure 1 Transthoracic echocardiogram in the subcostal view with color Doppler
fl ow imaging showing the large left to right shunt of a secundum atrial septal defect.Figure 2 Cardiac computed tomography showing the large secundum atrial septal
defect (green arrow) as well as the dilated right atrium (RA) and right ventricle (RV); LA (left atrium), LV (left ventricle).
Figure 3 Curved multiplanar reformat of computed tomography coronary angi-
ography demonstrating the anomalous left main coronary artery (green arrows) arising from the right coronary cusp then traversing between the aorta and the pulmonary trunk before bifurcating into the left anterior descending and circum fl ex
arteries. LA (left atrium), LV (left ventricle).
Figure 4 Three-dimensional reconstructed, volume rendered computed tomogra-
phy coronary angiography showing the course of the anomalous left main coronary artery (green arrows) arising from the right coronary cusp then traversing between the aorta and right ventricular out fl ow tract (RVOT).
| 12439b7452d8cc28f773b2e3fbe7a31ebaebff94 | page_0001 |
Vascular Health and Risk Management 2008:4(1) 261Anomalous left main coronary artery and an isolated secundum atrial septal defect
A transthoracic echocardiogram obtained to further
evaluate the murmur revealed normal left ventricular systolic function and hyperdynamic right ventricular systolic function with normal wall motion. Left ventricular and left atrial sizes were normal, but there was right ventricular and right atrial dilatation along with right ventricular volume overload. A large secundum atrial septal defect (ASD) with left to right shunt was evident (Figure 1) with a calculated pulmonary-to-systemic blood fl ow ratio
(Qp/Qs ) estimated at 2.3 to 1.
His symptoms of “choking” with exertion, however,
were not typical presenting symptoms in an isolated ASD. As such, a right and left heart catheterization were performed to further evaluate his right sided heart pressures, shunt fraction and coronary anatomy. Pulmonary artery pressure was 29/9 mmHg (mean 16 mmHg) with a mean right atrial pressure of 6 mmHg. Mixed venous saturation was 73%, right atrial saturation 85% and pulmonary artery saturation 96%. Qp/Qs was calculated to be 4.60.
Subsequent coronary angiography demonstrated a right dominant system with a normal appearing right coronary artery. The left main coronary artery was found to anomalously originate from the anterior right coronary cusp. The left main, left circumfl ex and left anterior descending
artery appeared to be angiographically normal; however, we were not able to reliably determine its anatomic course in relation to the great vessels.
Because of the potential high-risk of sudden cardiac
death in patients with an anomalous left main coronary artery (ALMCA), we felt it was prudent to completely defi ne the
route of the left main coronary artery in relation to the great vessels as an inter-arterial course between the aorta and pulmonary artery would warrant surgical intervention. As such, a 64-slice multidetector computed tomography coronary angiography (CTCA) was performed to further evaluate the coronary anatomy and ASD. The CTCA demonstrated a large ASD along with a dilated right ventricle and right atrium (Figure 2). Qp/Qs was 2.3 by CTCA using left and right
ventricular stroke volumes. The left main coronary artery was seen to arise from the right coronary cusp then traverse between the pulmonary trunk and the proximal ascending aorta before bifurcating into the left anterior descending and circumfl ex arteries, which then followed their normal courses
distally (Figures 3 and 4). All vessels were patent with no critical coronary artery stenoses.
The risk of death from an ALMCA coursing between the
aorta and the pulmonary trunk is diffi cult to evaluate as most
cases are fi rst recognized at autopsy and are rarely suspected or identifi ed during life (Frescura et al 1998; Basso et al
2000). Most estimates of risk for sudden cardiac death have ranged from 36%–59% (Angelini et al 2002; Moustafa et al 2007). Basso and colleagues (2000) suggest that operation is probably mandatory for these patients. Given this high risk of sudden cardiac death, the patient underwent surgical re-implantation of the ALMCA to the left coronary cusp and repair of the ASD. He was discharged home after an uneventful hospital course, and on follow-up is doing well with complete resolution of his symptoms.
ASD is the second most common form of congenital heart
disease found in adults (30%) with the secundum type being the most common (75%) (Wu and Child 2004). The incidence of ALMCA from the right coronary cusp is rare in the general population (0.02%) and one of the less frequent types of anomalous coronaries (1.3%) (Y amanaka and Hobbs 1990). Anomalous coronary arteries are more commonly associated with other congenital heart defects like tetralogy of Fallot, transposition of the great arteries, double outlet right ventricle, univentricular hearts, and truncus arteriosus (Vlodaver et al 1975). There have been case reports of other coronary anomalies in patients with an ASD such as an anomalous right coronary artery (Maki et al 2001) and anomalous left coronary artery from pulmonary artery (Turley et al 1995; Schneider et al 2006); however, to our knowledge this is the fi rst reported case of an isolated secundum ASD and ALMCA
with an inter-arterial course in the same patient. CTCA is a well recognized and appropriate modality for the evaluation of suspected anomalous coronary arteries (Hendel et al 2006) and has a high negative predictive value for excluding critical coronary artery stenoses (Achenbach 2007). It may also be a valuable tool in patients with ASDs for further evaluation of concomitant cardiac pathology, shunt calculation and coronary stenoses prior to referral for defi nitive therapy.
Disclosure
The authors report no confl icts of interest.
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043109-6 Loas, Alouini, and Vallet Rev. Sci. Instrum. 85, 043109 (2014)
equal to 9 mrad, the measured signal never exceeds 1.2 μrad.
The drastic reduction of this systematic effect is made possi-
ble because the sensing beams are depolarized. This justifiesthe need of perfectly depolarized beams and consequently the
use of a passive interferometer.
Measurements on chiral compounds can be now con-
ducted. To compare with previous measurements, we con-
sider samples formerly tested at 488 nm with an active
interferometer
5and at 633 nm with passive interferometers.6
However, a rough λ−2dependence for the magnetochiral in-
dex is predicted, both from dipole-dipole interaction model31
and from full quantum-mechanical considerations.32The ex-
pected effect is thus lower at 1550 nm compared to visi-
ble wavelengths and a large rotatory dispersion is requiredin order to get a detectable magnetochiral effect. Three
molecules comply pretty well with the previous requirements:
limonene, 3-(trifluoroacetyl)-camphor, and carvone. Thesethree molecules are fairly transparent at 1550 nm, both enan-
tiomers are available and the optical activity is rather large.
The first measurement was done with racemic limonene. Asystematic phase shift of 30 nrad was detected. We have then
successively replaced the racemic mixture by R( +)-limonene
and S( −)-limonene. Even with large integration times
(1000 s) on the lock-in amplifier, corresponding to a noise
rejection equivalent bandwidth /Delta1f=0.1 mHz, we did not de-
tect any magnetochiral directional phase shift within 10 nrad
fluctuations.
By considering that a signal-to-noise ratio of 3.3 avoids
false alarm and false dismissal (probability better than
99.6% for a Rayleigh distribution), we can thus affirm that
/Delta1φ
MC<33 nrad. Defining the magnetochiral index as nMC
=(/Delta1φ MCλ)/(4πLC), it yields a limit nMC<4×10−13T−1.
Similar negative results were obtained on carvone and 3-
(trifluoroacetyl)-camphor.
Assuming absorption bands in the far UV , an order of
magnitude of the expected phase shift /Delta1φ MCcan be obtained
from an expression derived from the Bequerel relation ex-tended to magnetochiral interaction
10,29
/Delta1φMC=eH
2πmcλλ2
0/parenleftbig
λ2−λ2
0/parenrightbig2θA, (12)
where e and m are the charge and mass of the electron and
where the angle of rotation due to optical activity obeys
the following wavelength dependence θ(λ)=θA/(λ2−λ2
0).
For Limonene, θAandλ0are estimated to be, respectively,
600◦/dm and 210 nm. Equation (12) then leads to /Delta1φ MC
=500 nrad, that is, nMC=4.7×10−11T−1at 1550 nm. This
is two orders of magnitude above the noise floor of our instru-
ment. This can also be compared to the values measured at
visible wavelength. For limonene, we measured in the past6
nMC=3.9(±1.3)×10−10T−1at 488 nm, while at 633 nm,
Kleindienst and Wagnière5measured, for 3-(trifluoroacetyl)-
camphor and for carvone, respectively, nMC=3(±0.2)×10−8
T−1andnMC=1.3(±0.3)×10−9T−1.A g a i n ,a λ−2depen-
dence for the magnetochiral index then leads to expected es-
timations at 1.55 μm two orders of magnitude above detec-
tion limit of our instrument. These unexpected results might
mean that the λ−2dependence has to be reconsidered or/andthat the magnetochiral index is actually much lower than that
expected from Bequerel model. Further theoretical investiga-
tions are thus required. For instance, we have assumed thatoptical index is only due to UV transitions.
31Contributions
to residual absorption bands in the visible or infrared should
modify the expected value. Furthermore, at 1550 nm, contri-butions to IR vibrational transitions have probably to be taken
into account.
V. CONCLUSION
An apparatus combining a depolarized fiber-optic Sagnac
interferometer and a double modulation-demodulationscheme has been designed to measure the non-reciprocal
phase shifts associated to the magnetochiral index. We have
validated a depolarization level better than 10
−5. This permits
to decrease the amplitude of systematic phase shifts, mainly
due to residual birefringences of the cell windows, below
30 nrad. The good stability of the interferometer allows oneto reach a measurement time of 1000 s with a noise-floor
value of 0.5 μrad/Hz
1/2comparable with the state-of-the-art.
This yields an experimental detection level of 33 nrad witha confidence level of 99.6%. Tests on three different organic
molecules have shown that the magnetochiral index is lower
than 4 ×10
−13T−1at 1.55 μm. As compared to figures
previously obtained for the same molecules in the visible,5,6
it implies a significant discrepancy with the values expected
from a λ−2dependence for the magnetochiral index. To
validate our results, the next step is consequently to compute
accurately the magnetochiral index at 1.55 μm from ab initio
models.13
As the detection performance reaches the noise floor de-
fined by the relative intensity noise of the superluminescentsource, it seems difficult to drastically lower the limit of
this setup below 33 nrad, the acquisition time being already
set to its maximum. Increasing this acquisition time furtherwould only slightly diminish the noise floor, at the expense
of a larger sensitivity to thermal and mechanical long-term
fluctuations. In order to detect magnetochiral index with oursetup, a possibility would be to use larger alternated magnetic
field.
4Alternatively, the availability of samples that present
larger magnetochiral birefringence is an open question. In-
deed, compared to resonant passive ring interferometers,3,33
our single-pass setup is not sensitive to samples which present
residual absorption or diffusion. One can then consider using
samples made of chiral compounds mixed with ferrofluid,31
or chiral ferromagnets.11Organic compounds with a large
optical activity in the infrared, due to, e.g., delocalized π-
electrons such as helicene, could also be tested.34The appara-
tus could also be easily modified to test in-loop samples underreflexion at non-normal angle. This would permit to test solid
samples, e.g., photonics crystals
35where giant magnetochiral-
ity is expected. Perspective for the setup would also includethe extension to the detection of other magnetical directional
anisotropies in crystals.
36
ACKNOWLEDGMENTS
The authors thank Elsa Rescan for preliminary works,
Cyril Hamel and Ludovic Frein for technical assistance, | 10.1063_1.4871988 | page_0006 |
043109-7 Loas, Alouini, and Vallet Rev. Sci. Instrum. 85, 043109 (2014)
Thierry Ruchon and Jeanne Crassous for fruitful discussions.
This work was partly funded by Université de Rennes 1.
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pdf.
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GmbH&Co., Hanau, Germany, 2014). See http://heraeus-quarzglas.com .
31T. Ruchon, Ph.D. thesis, Université de Rennes 1, France, 2005; the
manuscript (in French) can be downloaded from http://tel.archives-ouvertes
.fr/tel-00541349 .
32L. D. Barron, Molecular Light Scattering and Optical Activity , 2nd ed.
(Cambridge University Press, Cambridge, UK, 2004).
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Appl. Phys. Lett. 88, 113105 (2006); https://doi.org/10.1063/1.2185447 88, 113105
© 2006 American Institute of Physics.Observation of low-lying excitations of
electrons in coupled quantum dots
Cite as: Appl. Phys. Lett. 88, 113105 (2006); https://doi.org/10.1063/1.2185447
Submitted: 22 November 2005 • Accepted: 13 February 2006 • Published Online: 15 March 2006
César Pascual García , Sokratis Kalliakos , Vittorio Pellegrini , et al.
| 10.1063_1.2185447 | page_0000 |
Observation of low-lying excitations of electrons in coupled quantum dots
César Pascual García,a/H20850Sokratis Kalliakos, and Vittorio Pellegrini
NEST CNR-INFM and Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126 Pisa, Italy
Aron Pinczuk
Bell Laboratories and Lucent Technologies, Murray Hill, New Jersey and Department of Physics and
Department of Applied Physics and Applied Mathematics, Columbia University, New York, New York 10027
Brian S. Dennis, Loren N. Pfeiffer, and Ken W. West
Bell Laboratories and Lucent Technologies, Murray Hill, New Jersey 07974
/H20849Received 22 November 2005; accepted 13 February 2006; published online 15 March 2006 /H20850
Tunneling excitations of electrons in dry-etched modulation-doped AlGaAs/GaAs coupled quantum
dots /H20849QDs /H20850are probed by resonant inelastic light scattering. A sequence of intra- and intershell
excitations are found at energies determined by the interplay between the QD confinement energy/H6036
/H92750and the tunneling gap /H9004SAS, the splitting between the symmetric and antisymmetric delocalized
single particle molecular states. The narrow linewidths displayed by electronic excitations in thesenanostructures indicate promising venues for the spectroscopic investigation of entanglement ofelectron states in these artificial molecules. © 2006 American Institute of Physics .
/H20851DOI: 10.1063/1.2185447 /H20852
Semiconductor quantum dots /H20849QDs /H20850are regarded as ar-
tificial atoms with electron properties that can be tailored ondemand.
1In this framework, artificial molecules can be real-
ized with two interacting QDs.2It has been proposed that
coupled QDs are of particular relevance for quantum com-putation schemes.
3,4In fact, the tunneling gap /H9004SASthat splits
the QD levels into their symmetric and antisymmetric com-binations can modulate the exchange interaction and pro-vides a route towards entanglement of two spins.
5Electron
states in coupled double QDs are expected to be less sensi-tive to dephasing mechanisms linked to coupling of spin andcharge states that occur in a single QD environment. CoupledQDs also offer ways to study novel spin and charge collec-tive phases and quantum phase transitions at the nanoscale.
6
The remarkable physics and applications of coupled
QDs is manifested in several experimental studies. Transportexperiments based on tunneling have extensively investi-gated interdot coupling effects in the strong and weak cou-pling regimes.
7–9These measurements have provided evi-
dence for the impact of symmetric and antisymmetric statesin the Coulomb blockade spectra and have demonstrated
slower relaxation rates for spin states in laterally coupledQDs and their coherent manipulation. Interband lumines-cence experiments have also been carried out in self-assembled InAs coupled QDs.
10
In this letter, we report the observation of low-lying neu-
tral excitations of electrons in vertically coupledGaAs/AlGaAs double QDs nanofabricated by e-beam li-thography and dry etching. Resonant inelastic light scatteringspectra access excitations linked to the tunneling and quan-tum confinement degrees of freedom of electrons. The spec-tra reveal sharp excitations, with a full width at half maxi-mum /H20849FWHM /H20850below 200
/H9262eV, that demonstrates the high
quality of these etched nanostructures. These QD systemscould be suitable for exploration of entanglement of electronstates that enter in quantum computation schemes based onthe optical manipulation of spin and charge.Eigenstates of a parabolic QD in the noninteracting
scheme are described by the Fock-Darwin /H20849FD/H20850levels. In
vertical QDs, the coupling between the two dots leads to thesplitting of the single dot levels in bonding /H20849symmetric /H20850and
antibonding /H20849antisymmetric /H20850levels separated by the tunnel-
ing gap /H9004
SAS. The single particle eigenenergies of parabolic
double QDs at zero magnetic field can thus be modeled bythe following equation:
E
N,P=/H6036/H92750/H208492n+/H20841m/H20841+1/H20850+P/H9004SAS=/H6036/H92750/H20849N+1/H20850
+P/H9004SAS, /H208491/H20850
where n=0,1,..., m=0±1,... are the radial and azimuthal
quantum numbers, respectively, N=2n+/H20841m/H20841identifies shells
with well-defined atomiclike parities, /H6036/H92750is the confinement
energy, and Pis the extra degree of freedom labeled as a
pseudospin. Ptakes values −1/2 for symmetric levels and
1/2 for antisymmetric levels. This peculiar energy-levelstructure yields an excitation spectrum characterized by tun-neling or pseudospin modes constructed from intra- or inter-shell excitations of electrons from the symmetric to the an-tisymmetric QD levels. Figure 2 /H20849a/H20850shows the FD sequence
following Eq. /H208491/H20850in case /H9004
SASis slightly larger than /H6036/H92750.
Parity selection rules applied to parabolic double
coupled QDs establish that monopole transitions with/H9004m=0/H20849/H9004N=0,2,4,... /H20850and/H9004P=0, ±1 are the strongest in-
tensity modes active in light scattering experiments in a
backscattering geometry.11,12The single-particle representa-
tions of intra- and intershell pseudospin excitations associ-ated with changes of NandPare shown in the left panel of
Fig. 2 /H20849a/H20850as vertical arrows. In agreement with this scheme,
we found in our experiments two lowest-energy peaks in thelow-temperature inelastic light scattering spectra at energiesbelow 1 meV that were assigned to the intrashell /H9004P=1 ex-
citation at /H9004
SASand to the intershell pseudospin mode at
2/H6036/H92750−/H9004SAS. These peaks are remarkably sharp with a
FWHM of 200 /H9262eV and allow direct measurement of
/H9004SAS=1±0.1 meV and /H6036/H92750=0.8±0.05 meV in our system.
Consistent with this assignment, we found a broader inter-a/H20850Electronic mail: [email protected] PHYSICS LETTERS 88, 113105 /H208492006 /H20850
0003-6951/2006/88 /H2084911/H20850/113105/3/$23.00 © 2006 American Institute of Physics 88, 113105-1 | 10.1063_1.2185447 | page_0001 |
shell monopole mode at2 /H6036/H92750=1.8±0.1 meV. Contrary to
the case of single QDs, this intershell excitation has amarked temperature dependence and disappears around15 K. The measured activation gap is consistent with thevalue of 2 /H6036
/H92750−/H9004SAS, offering further evidence of the impact
of interdot coupling on the excitation spectrum. Finally, anadditional broad mode is observed around 2.4±0.4 meV cor-responding to 4 /H6036
/H92750−/H9004SAS.
The sample was fabricated starting from an 18 nm wide,
symmetrically modulation-doped Al 0.1Ga0.9As/GaAs double
quantum well with 6 nm Al 0.1Ga0.9As barrier separation
/H20849layer sequence and conduction band profile along the
growth direction are shown in Fig. 1 /H20850. The tunneling gap
measured by light scattering in the double layers /H20849data not
shown /H20850is 0.8 meV. The low-temperature electron density
and mobility measured from Shubnikov–de Haas are ne=3
/H110031011cm−2and 2.7 /H11003106cm2/V s, respectively. The lateral
confinement was produced by inductive coupled plasma re-active ion etching /H20849ICP-RIE /H20850. To this end, a 30 nm thick
nickel mask was first deposited on top of the sample.Coupled QD arrays /H20849with sizes 100 /H11003100
/H9262m2containing
104single coupled QD replica /H20850with lateral dot diameters of
about 400 nmwere defined by electron-beam lithography.This value of lateral diameter was chosen to yield a confine-ment energy of the order of the tunneling gap and a largeelectron occupation /H20849above 100 /H20850. In this many-electron case,
in fact, the light scattering response at resonance is domi-nated by single-particle transitions linked to Eq. /H208491/H20850and not
corrected by dynamical many-body effects.
11Deep etching
/H20849below the doping layer /H20850was then achieved by using a mix-
ture of BCl 3/Cl2/Ar in the ICP-RIE and applying low volt-
ages. The nickel mask was removed before the optical stud-ies. Top panels in Fig. 1 show scanning electron microscope/H20849SEM /H20850images of the coupled dots in the array. Light scatter-
ing experiments were performed in a backscattering configu-ration /H20849q/H333552/H1100310
4cm−1where qis the wave vector trans-ferred into the lateral dimension /H20850with temperatures down to
T=1.9 K. A tunable ring-etalon Ti:sapphire laser was used
and the scattered light was collected into a triple gratingspectrometer equipped with a charge-coupled device /H20849CCD /H20850
detector.
Right panel of Fig. 2 shows the resonant inelastic light
scattering spectra at different excitation energies andT=1.9 K after conventional subtraction of the background
due to interband luminescence. The laser with intensity of0.1 W/cm
2was tuned between 1528.6 meV /H20849bottom spec-
trum /H20850to 1534.5 meV /H20849top spectrum /H20850to explore different
resonances. The spectra were taken in a depolarized configu-ration with perpendicular polarizations of the incoming andoutgoing light in order to reduce the stray laser light.
Spectra shown in Fig. 2 are remarkably different from
those found in single QDs with the same lateral diameter.
13
In single QDs the resonant inelastic light scattering spectradisplay a sequence of peaks equally spaced in energy by2/H6036
/H92750with a FWHM of 1 meV in agreement with data ob-
tained by other groups.11The spectra of double QDs are
instead characterized by two sharp /H20849FWHM=0.2 meV /H20850low-
energy peaks at 2 /H6036/H92750−/H9004SASand/H9004SAS. The peak at 1.8 meV
corresponding to 2 /H6036/H92750is thus assigned to the conventional
intershell /H9004N=2 mode also observed in the single QDs. The
peculiar energy level structure of the coupled QDs is addi-tionally revealed by the fourth highest-energy peak shown inFig. 2 and observed at an energy corresponding to4/H6036
/H92750−/H9004SAS=2.4 meV. The energy of this peak shifts from
2.2 to 2.6 meV depending on the laser excitation wave-length. This behavior can be linked to nonparabolicity effectswhose impacts increase with the energy of the mode and topartial overlap with the 2 /H6036
/H92750+/H9004SASintershell pseudospin
mode expected at /H110112.8 meV. More excitations were de-
tected at higher energies /H20849data not shown /H20850with decreasing
intensity and with broader signal corresponding to higherintershell excitations combined with the symmetric and anti-symmetric states. It can be noted that the energy of the in-trashell pseudospin mode /H20849/H9004
SAS=1 meV /H20850is higher than the
FIG. 1. Top: SEM images of one dry-etched QD and the 100 /H11003100/H9262m2
array composed by 104coupled QDs separated by 1 /H9262m. Bottom: Schematic
description of the vertical double QD and the corresponding energy diagramin the conduction band. The two lowest symmetric and antisymmetric levelsare also shown. Pis the pseudospin and /H9004
SASis the tunneling gap of the
coupled QD.
FIG. 2. Left panel: schematic representation of energy levels and transitionsin the coupled QD. NandPare the shell and pseudospin quantum numbers,
respectively. Black and grey lines represent symmetric and antisymmetriclevels, respectively. The dotted line marks the position of the Fermi levelthat accounts for the observed reduced intensities of the two lowest-energymodes. Right panel: Resonant inelastic light scattering spectra at 1.9 K andat different laser energies /H20849shown in the figure /H20850in depolarized configuration.
Intensities were scaled by factors indicated in the figure.113105-2 García et al. Appl. Phys. Lett. 88, 113105 /H208492006 /H20850 | 10.1063_1.2185447 | page_0002 |
single-particle excitation energy at the bare tunneling
gap measured in the double quantum well prior to nano-fabrication /H20849/H110110.8 meV /H20850. This is probably due to partial
depletion of electrons caused by the etching processes.
The difference in the intensities between the modes be-
low 1 meV and those at higher energies is remarkable. Itsuggests partial population of the two highest-energy occu-pied levels as indicated by the position of the Fermi levelshown by the dotted line in the left part of Fig. 2. These twolevels are the excited states associated with the two sharplow-lying transitions. Their partial population explains thereduced intensities of the two modes due to phase space fill-ing effects. The results in Fig. 2 therefore suggest that lightscattering can be applied to determine both energies andpopulation of molecular states in coupled QDs.
Further evidence of the impact of interdot coupling
arises from the temperature behavior of the conventional in-trashell monopole excitation at 2 /H6036
/H92750. Contrary to the single
QD case, where this excitation remains unchanged up totemperatures above 30 K, a significant change of the signalintensity occurs at much lower temperatures and the inter-shell mode disappears around 15 K with an activation energyof 0.8 meV as displayed in the inset to Fig. 3. The activationgap is consistent with the value of 2 /H6036
/H92750−/H9004SAS, the gap sepa-
rating the highest-energy occupied level /H20849an antisymmetric
state with shell number N/H20850from the lowest-energy unoccu-
pied level /H20849a symmetric state with shell number N+2/H20850. This
behavior thus offers further evidence of the impact of inter-dot coupling in the excitation spectrum.In conclusion, we reported the first measurements of ex-
citations of electrons in nanofabricated vertically coupledQDs. The spectra reveal a low-lying intrashell pseudospinmode across the tunneling gap as well as intershell excita-tions resulting from the interplay between the confinementenergy and the tunneling gap. The results presented here sug-gest that, by offering access to molecularlike excited states inthe coupled QDs, the light scattering methods can provide awealth of quantitative information on the energy level se-quence, level occupation, and tunneling gap in double QDs.Further work in the few-electron occupation regime shouldaddress the interplay between spin and pseudospin excita-tions in the coupled dots.
The authors acknowledge support from the Italian Min-
istry of Foreign Affairs, the Italian Ministry of Research/H20849FIRB-RBAU01ZEML /H20850, the European Community’s Human
Potential Programme /H20849HPRN-CT-2002-00291 /H20850, the National
Science Foundation /H20849Grant No. DMR-03-52738 /H20850, and the
Department of Energy /H20849Contract No. DE-AIO2-04ER46133 /H20850.
The authors are grateful to SENTECH-Berlin for allowing usto use the ICP-RIE machine. The authors acknowledge use-ful discussions with Massimo Rontani, Guido Goldoni, andElisa Molinari.
1S. M. Reimann and M. Manninen, Rev. Mod. Phys. 74, 1283 /H208492002 /H20850.
2V. G. Van Der Wiel, S. De Franceschi, J. M. Elzerman, T. Fujisawa,
S. Tarucha, and L. P. Kouwenhoven, Rev. Mod. Phys. 75,1/H208492003 /H20850.
3D. P. DiVincenzo, D. Bacon, J. Kempe, G. Burkard, and K. B. Whaley,
Nature /H20849London /H20850408, 339 /H208492000 /H20850.
4D. Loss and D. P. DiVincenzo, Phys. Rev. A 57,1 2 0 /H208491998 /H20850.
5G. Burkard, D. Loss, and D. DiVincenzo, Phys. Rev. B 59, 2070 /H208491999 /H20850.
6M. Rontani, S. Amaha, K. Muraki, F. Manghi, E. Molinari, S. Tarucha,
and D. G. Austing, Phys. Rev. B 69, 085327 /H208492004 /H20850.
7T. Hatano, M. Stopa, and S. Tarucha, Science 309, 268 /H208492005 /H20850.
8J. R. Petta, A. C. Johnson, J. M. Taylor, E. A. Laird, A. Yacoby, M. D.
Lukin, C. M. Marcus, M. P. Hanson, and A. C. Gossard, Science 309,
2180 /H208492005 /H20850.
9T. H. Wang and S. Tarucha, Appl. Phys. Lett. 71, 2499 /H208491997 /H20850.
10H. J. Krenner, M. Sabathil, E. C. Clark, A. Kress, D. Schuh, M. Bichler,
G. Abstreiter, and J. J. Finley, Phys. Rev. Lett. 94, 057402 /H208492005 /H20850.
11R. Strenz, U. Bockelmann, F. Hirler, G. Abstreiter, G. Böhm, and G.
Weimann, Phys. Rev. Lett. 73, 3022 /H208491995 /H20850; D. J. Lockwood, P. Hawry-
lak, P. D. Wang, C. M. Sotomayor Torres, A. Pinczuk, and B. S. Dennis,ibid. 77,3 5 4 /H208491996 /H20850; C. Schuller, K. Keller, G. Biese, E. Ulrichs, L. Rolf,
C. Steinebach, and D. Heitmann ibid. 80, 2673 /H208491999 /H20850.
12C. Pascual García, V. Pellegrini, A. Pinczuk, M. Rontani, G. Goldoni, E.
Molinari, B. S. Dennis, L. N. Pfeiffer, and K. W. West, Phys. Rev. Lett.
95, 266806 /H208492005 /H20850.
13C. Pascual García, V. Pellegrini, A. Pinczuk, M. Rontani, G. Goldoni, E.
Molinari, B. S. Dennis, L. N. Pfeiffer, and K. W. West, Physica E, in press.
FIG. 3. Temperature dependence of the monopole intershell transition at2/H6036
/H92750. Laser intensity and energy are 0.1 W/cm2and 1567 meV. Spectra
are presented after conventional subtraction of background due to lumines-cence. The inset shows an Arrhenius plot of the integrated intensity with anactivation energy of 0.8 meV.113105-3 García et al. Appl. Phys. Lett. 88, 113105 /H208492006 /H20850 | 10.1063_1.2185447 | page_0003 |
1
Scientific REPORTS | (2018) 8:6163 | DOI:10.1038/s41598-018-24499-5www.nature.com/scientificreportsStructure and Properties of DNA
Molecules Over The Full Range of
Biologically Relevant Supercoiling
States
Paolo Bettotti1, Valeria Visone2, Lorenzo Lunelli3,4, Giuseppe Perugin o2, Maria Ciaramella2 &
Anna Valenti2
Topology affects physical and biological properties of DNA and impacts fundamental cellular processes,
such as gene expression, genome replication, chromosome structure and segregation. In all organisms DNA topology is carefully modulated and the supercoiling degree of defined genome regions may
change according to physiological and environmental conditions. Elucidation of structural properties of
DNA molecules with different topology may thus help to better understand genome functions. Whereas a number of structural studies have been published on highly negatively supercoiled DNA molecules, only preliminary observations of highly positively supercoiled are available, and a description
of DNA structural properties over the full range of supercoiling degree is lacking. Atomic Force
Microscopy (AFM) is a powerful tool to study DNA structure at single molecule level. We here report a comprehensive analysis by AFM of DNA plasmid molecules with defined supercoiling degree, covering the full spectrum of biologically relevant topologies, under different observation conditions. Our data, supported by statistical and biochemical analyses, revealed striking differences in the behavior of
positive and negative plasmid molecules.
DNA topology is an intrinsic property of DNA molecules, and is controlled by the direct action of DNA topoi-
somerases
1–8. These are enzymes essential for proliferation and survival of all cells and are indeed important tar -
gets for chemotherapeutic drugs. In vitro , DNA topoisomerases are able to induce significant changes of linking
number (Δ Lk; see the Methods Section for definition of the topological parameters) of covalently closed DNA
molecules, by either removing or introducing supercoils. Each enzyme has its own specificity, and can modify the ΔLk stepwise, leading to production of a range of differently supercoiled molecules.
In vivo , genomic DNA is organized in topologically closed domains, whose supercoiling degree is strictly reg-
ulated, given the great impact of topology on all DNA activities
9,10. In organisms living in the range of mesophilic
temperatures, DNA is in general negatively supercoiled (− SC); thermophilic microorganisms, living in envi-
ronmental niches above 70 °C, possess a special DNA topoisomerase called reverse gyrase (RG), which is able to
catalyze positive supercoiling11–13. Recent results show that RG is essential for growth at 95 °C of a hyperthermo-
philic species, suggesting that the presence of the enzyme is necessary to maintain the correct DNA supercoiling in organisms living at high temperatures
14.
Positively supercoiled (+SC) DNA accumulates in every organism during DNA transactions, such as replica-
tion and transcription15; moreover, + SC DNA prevents extensive reversal of replication forks in the presence of
replication blocking lesions16, promotes telomere resolutions17 and marks centromeres as unique chromosome
loci18. In addition, condensin and cohesin complexes, which play essential role in chromosome architecture and
segregation during mitosis and meiosis, induce positive supercoiling19,20. Although current techniques do not
allow determination of supercoiling degree in vivo in real time, it is likely that the full range of topological degree
1Nanoscience Laboratory, Department of Physics, University of Trento, Via Sommarive 14, I-38123, Povo (Trento),
Italy. 2Institute of Biosciences and Bioresources, National Research Council of Italy, Via Pietro Castellino 111, 80131,
Napoli, Italy. 3Laboratory of Biomarker Studies and Structure Analysis for Health, FBK-Fondazione Bruno Kessler, Via
Sommarive 18, 38123, Povo, Trento, Italy. 4Institute of Biophysics, National Research Council of Italy, Trento, Italy.
Paolo Bettotti, Valeria Visone and Lorenzo Lunelli contributed equally to this work. Correspondence and requests for
materials should be addressed to M.C. (email: [email protected]) or A.V. (email: [email protected])Received: 6 December 2017
Accepted: 4 April 2018
Published: xx xx xxxxOPEN
| 10.1038_s41598-018-24499-5 | page_0000 |
www.nature.com/scientificreports/2
Scientific REPORTS | (2018) 8:6163 | DOI:10.1038/s41598-018-24499-5may occur during the cell life and DNA supercoiling may vary significantly over the cell cycle, in distinct genome
locations as well as in response to environmental stimuli. Thus, understanding how DNA is organized over the full range of supercoiling degree may provide crucial insights into the principles that underlie genomic organiza-tion and regulation in living organisms.
So far, the structure of − SC plasmids has been investigated by several techniques
21,22. Electron (SEM, TEM)
and scanning probe microscopy (SPM) are the de-facto techniques to visualize DNA at single molecule level
and to unveil structural details. All these techniques suffer from possible artifacts during sample preparation, which might influence the measurements outcome
23–26. Atomic force microscopy (AFM) may in part overcome
these limitations, as it allows to control several parameters during plasmids deposition (e.g. buffer ionic strength, temperature, etc.) and to simulate in-vivo conditions by performing measurements in liquids. Although many structural studies have been reported on − SC plasmids, little information is available for + SC plasmids. Only
recently two papers reported the first observations at single molecule level of + SC DNA molecules, giving impor -
tant impulse to the field
27,28. However, further analyses are required to extend these results and fill important
gaps. Indeed, Irobalieva et al . used cryo-tomography to analyze DNA minicircles of a few hundred base pairs with
defined degrees of supercoiling. Although these molecules are useful models, they can only accommodate a few supercoils, due to their short size. More recently, Li et al . analyzed by AFM + SC plasmids and measured different
lengths between positive and negative topoisomers, whose Δ Lk was however undefined. These measurements
rely on few molecules only and their conclusions might be of limited relevance because of the reduced ensemble investigated
28.
In the present work, we present a systematic AFM analysis of DNA plasmids with ΔLk covering a wide range
of supercoiling (ranging from − 12 to + 12). Observations were performed under a number of different condi-
tions, and data are supported by quantitative and biochemical analysis, highlighting significant differences in the behavior of −SC and +SC molecules. The relevance of these results on the biological properties of DNA is
discussed.
Results and Discussion
Effect of immobilization conditions on structural conformation of plasmid molecules with dif -
ferent supercoiling degree observed by AFM. RG is a powerful tool to obtain plasmid molecules with
different degree of supercoiling: indeed, by changing reaction parameters (temperature, time, DNA/enzyme ratio and so on) it is possible to modulate the enzyme activity and obtain distinct populations of topological isomers with defined ΔLk (called topoisomers; see Fig. 1A for a scheme of the reaction)
29,30. Reactions were set up using
the 3.000 bp pBlueScript plasmid (pBs), purified from Escherichia coli cultures in its negatively supercoiled form,
and the RG from the archaeon Sulfolobus solfataricus10–12,29. In a time course experiment, aliquots of products
were withdrawn after different incubation times and analysed by two-dimensional gel electrophoresis, enabling separation of − SC and + SC topoisomers (Fig. 1A). Figure 1B shows supercoiling modification of the pBs plas-
mid with time, from − SC (time 0) to + SC (300 sec.). The topological state of each topoisomer population was
expressed calculating the mean specific linking number (mean σ ) obtained from the σ values of the most abun -
dant topoisomers produced in each reaction (see Methods). The mean σ of each topoisomer population was
plotted as a function of time, showing the increase of plasmid supercoiling density during reverse gyrase reaction (Fig. 1C).
Among the time course products, four plasmid populations with defined mean σ (−0.04; +0.01; +0.03;
≥ +0.04) were selected, purified and analysed by AFM in air. Since the only images available so far for positive
plasmids were obtained on silanized mica
28, it is not known whether these molecules are affected by the deposi -
tion procedure, prompting us to explore different deposition methods. In order to increase the mica affinity for negatively charged molecules such as DNA, different strategies can be followed. Cation-assisted deposition is widely used due to the simplicity of sample preparation; it has been suggested that nucleic acids deposition on mica in the presence of divalent cations results in relatively weak binding and allows for the equilibration of mol-
ecules during deposition. A further drawback of this technique is that the cation requirement limits the range of
experimental conditions that can be used. Alternatively, treatment of mica with aminosilanes is also an effective procedure for nucleic acids binding; silanized mica enables a stronger binding of nucleic acids and tends to trap DNA molecules in a configuration that corresponds to the 2D projection of their solution conformation
31,32; how -
ever, hydrolysis of aminosilanes and aggregation is a complication associated with this technique.
We tested both procedures and explored a wide range of experimental conditions to find deposition protocols
yielding reproducible binding of plasmids while preserving the specific features of molecules with different super -
coiling degree. We selected appropriate conditions for deposition on mica either in the presence of MgCl2 or after
treatment with 3-Amino propyl-trimethoxysilane (APTMS).
When deposited on APTMS-treated mica in the presence of 100 mM NaCl, highly −SC plasmids ( σ = −0.04)
assumed a plectonemic form with several crossings seen in each molecule and, occasionally, irregularly balled-up or aggregated forms (Fig. 2A). These shapes were already reported for other plasmids having a similar super -
coiling degree
33. In contrast, the populations of topoisomers with an intermediate degree of supercoiling
(+0.01 < mean σ < +0.03) lost the plectonemic form and showed a reduced number of crossings. Finally, when σ
reached the value of +0.04, the molecules adopted again a highly plectonemic conformation (Fig. 2A).
Strikingly different results were obtained when the same samples were deposited in the presence of MgCl2.
Highly −SC plasmids adopted a loose geometry, looking like open circular molecules or showing very few crosso-
vers (Fig. 2B), similar to those reported by Lyubchencko and colleagues34. A clear transition from relaxed to plec-
tonemic structures was observed at σ value of + 0.04, as seen for the same molecules deposited on APTMS-treated
mica. Thus, under these conditions the plectonemic aspect seems a peculiar feature of + SC molecules with a
mean σ ≥ +0.04. | 10.1038_s41598-018-24499-5 | page_0001 |
www.nature.com/scientificreports/3
Scientific REPORTS | (2018) 8:6163 | DOI:10.1038/s41598-018-24499-5Extensive comparison of the shapes of highly + SC molecules on the two surfaces showed that they appear
highly homogeneous (Fig. 3), thus suggesting that the shape of these molecules is not heavily affected by the dep-
ositions conditions. Moreover, these images show that the end-point product of RG is a homogeneous population
comprised of a single, or a few, topoisomer(s) with very close ΔLk.
To better understand the reasons of this striking difference, we acquired AFM images of linearized DNA
plasmids on APTMS-treated-mica and on bare mica in the presence of MgCl2 (data not shown) and meas-
ured their contour length. The measured contour length of our DNA molecules on APTMS-treated-mica was 985 ± 40 nm, in very good agreement with the estimated length of this 3000 bp long DNA molecule in B form,
which is 980.8 nm. In contrast, the experimental value obtained when DNA was deposited in the presence of Mg
Figure 1. Reverse gyrase time course reaction analysed by 2D-gel electrophoresis. (A) Schematic
representation of 2D-gel migration of −SC and +SC topoisomers. (B) −SC pBs DNA was incubated with S.
solfataricus RG30 at 90 °C for the time indicated and subjected to 2D electrophoresis. For each time point, the
mean σ was determined as described in Experimental Procedures. Representative gel is shown. (C) The graph
shows increasing of positive supercoiling (expressed as mean σ) with time during RG reaction. The curve is not completely linear, due to the complexity of the reaction, which is affected by a multiplicity of parameters
30,31.
Data are from three independent experiments. | 10.1038_s41598-018-24499-5 | page_0002 |
www.nature.com/scientificreports/4
Scientific REPORTS | (2018) 8:6163 | DOI:10.1038/s41598-018-24499-5cations was of 893 ± 60 nm, with a decrease of about 9%. In a recently published paper, Lipiec et al .32 suggested
that the local structure of DNA molecules deposited on aminosilane-treated mica resembles that of the B crystal
form; in contrast, when molecules were deposited using the divalent cation adhesion method, a partial transition toward the A form occurs. If we take into account the structural parameters of the two DNA forms (A form: twist 32.7 degrees, rise 0.26 nm per base. B form: twist 34.3 degrees, rise 0.34 nm per base)
35 it is possible to estimate the
amount of B to A transition, by comparing the contour length of linear DNA in the two deposition conditions31.
Figure 2. AFM imaging of distinct topoisomer populations deposited on mica under different conditions. (A)
APTMS-treated mica or (B) in the presence of MgCl2. Aliquots of DNA were withdrawn during RG reaction at
indicated reaction time and analyzed by AFM. Insets show the migration on 2D gel of each purified population at time 0 (σ = −0,04), after 150” (σ = +0,01), 290” (σ = +0,03) and 300” (σ ≥ +0,01) of reaction. The RG
reaction products were monitored on 650 plasmids (about 65 plasmids for each timepoint). | 10.1038_s41598-018-24499-5 | page_0003 |
SURGERY ARTICLES
Clinical diagnosis and wide-awake surgical treatment
of proximal median nerve entrapment at the elbow:
a prospective study
Elisabet Hagert
Published online: 4 January 2013
#American Association for Hand Surgery 2013
Abstract
Background Proximal median nerve entrapment (PMNE, or
pronator syndrome) at the elbow has traditionally been
considered an elusive and rare diagnosis, as it is seldom
detectable using electrophysiological techniques. In this pa-per, the clinical manifestations, physical diagnosis, surgical
technique, and results of surgical treatment of PMNE are
presented, with accompanying instructional video.
Patients/Methods During 2011, 44 patients with PMNE
were surgically released and followed prospectively, 22
women/22 men, mean age 48.8 (range 25 –66). The patients
were equally distributed between right/left hands (23/21)
and the dominant hand was treated in 56 % of cases. The
diagnosis was based on: (1) weakness in median innervatedmuscles distal to the lacertus fibrosus; (2) pain upon pres-
sure over the median nerve at the level of the lacertus
fibrosus; and (3) positive scratch collapse test. A minimallyinvasive surgical treatment using only local anesthesia with
lidocaine –epinephrine and no tourniquet was used, and di-
rect perioperative return of strength in median innervatedmuscles was seen in all subjects.
Results The average preoperative quick DASH was 35.4
(range 6.8 –77.2); work DASH, 44.3 (6.25 –100); and activ-
ity DASH, 61.6 (12.5 –100). There were 71.1 % patients
who completed the 6-month follow-up, and the average
postoperative quick DASH was 12.7 (range 0 –43.1), which
is a statistically significant reduction ( p<0.0001; Student ’s
paired t test). Similarly, the work and activity DASH wassignificantly reduced ( p<0.001) to 12.5 (0 –75) and 6.25
(0–50), respectively.
Conclusions PMNE at the level of the lacertus fibrosus
should be called lacertus tunnel syndrome to distinguish it
from other levels of median nerve entrapment. It is a clinicaldiagnosis based on three distinct clinical findings: weak-
ness, pain over point of compression, and positive scratch
collapse test. Surgical release in local anesthesia allows for asafe, ambulatory, and cost-efficient procedure with low
morbidity.
Keywords Entrapment .Median nerve .Pronator
syndrome .Surgery .Wide-awake
Background
The pronator syndrome, or proximal median nerve entrap-
ment (PMNE) at the level of the elbow, was first describedin literature in the 1950s [ 18,22], but unlike its distal coun-
terpart —the carpal tunnel syndrome (CTS) —there are only
around 50 publications on the diagnosis and treatment ofPMNE, whereas a PubMed search on CTS will result in
>7,500 publications. This disparity has led surgeons to
claim that PMNE is an “extremely rare diagnosis ”[16]
and a “diagnosis of belief or disbelief ”[15], as objective
measurements of PMNE can seldom be retrieved.
In what lies the elusiveness of this diagnosis? In this age
of insurance-driven medicine, the dependency on so-called
objective measurements, i.e., electromyography (EMG) and
nerve conduction studies (NCS), is brought to the forefront
of medicine whereas clinical diagnosis and examination is
often considered subjective, biased, and unreliable [ 8]. In
the case of PMNE, however, there is a conundrum in that
proximal median EMG/NCS often is normal or inconclu-
sive, may simulate CTS [ 5], and has an unreliable specificityElectronic supplementary material The online version of this article
(doi:10.1007/s11552-012-9483-4) contains supplementary material,
which is available to authorized users.
E. Hagert ( *)
Department of Clinical Science and Education, Hand & FootSurgery Center, Karolinska Institutet, Storängsv.10,
11542 Stockholm, Sweden
e-mail: [email protected] (2013) 8:41 –46
DOI 10.1007/s11552-012-9483-4 | 515a2f826486c30c068d75b1dc5c0dd1d8277373 | page_0000 |
of 30 –70 % [ 3]. The shortcomings of electrodiagnostic
studies lies in the fact that a nerve may be compressed and
cause symptoms distal to the level of compression due tochanges in axonal transport and intraneural circulation
[17], but the pressure levels of compression are too low
to actually cause axonal injury [ 7] and thus no visible
changes in EMG/NCS. More recently, magnetic reso-
nance imaging (MRI) has been suggested as a potential
aid in proximal entrapment diagnosis, but in the case ofPMNE, the MRI findings are usually normal until there
is evident axonal degeneration and muscle wasting of
the forearm [ 1].
The objective of this manuscript is to revisit the clinical
findings and diagnosis of PMNE, as well as describe an in
situ approach to surgical decompression of the median nerveat the elbow, with video guidance.
Clinical Diagnosis and Treatment
Already in 1874, Erb emphasized the importance of clinical
diagnosis for determination of level of nerve affliction in the
upper extremity [ 2]. Similarly, the earliest publications on
PMNE describe the need of thorough physical examinationto diagnose the patient [ 18], in particular the presence of
weakness in muscles innervated by the median nerve both in
the hand and the forearm. The efficacy (sensitivity andspecificity) of manual muscle testing of antagonist muscles
to determine levels of focal neuropathies in the upper ex-
tremities have been established in randomized, blinded va-lidity studies to 88 –93 %, respectively [ 11,12].
The principle is easy: distal to the level of nerve afflic-
tion, distinct patterns of muscle weaknesses will be clinical-ly present. In the case of PMNE, these patterns have been
determined to primarily correlate to weakness in the flexor
pollicis longus (FPL), flexor digitorum profundus II (FDP
II), and the flexor carpi radialis (FCR) [ 19,20].
Symptoms and Clinical Findings
The most common complaints in patients with PMNE at the
level of the lacertus fibrosus is a loss of key and tip pinchstrength, a loss of fine motor skills and sense of clumsiness
(dropping objects), and, rarely, transient paresthesias in the
median nerve innervated region of the hand (thumb-radial
aspect of ring finger) [ 10].
Clinically, a symptomatic triad will be evident: (1) the
patient will have distinct weakness when manually testing
the strength of the muscles innervated by the median nerve
distal to the lacertus fibrosus, especially the FPL, FDP II,and FCR (see Video 2); (2) external pressure of the median
nerve at the level of the lacertus fibrosus will elicit distinct
pain and, at times, a positive Tinel ’s sign; and (3) a positivescratch collapse test [ 6] over the median nerve at the level of
the lacertus fibrosus (see Video 1).
Surgical Decompression
Surgical treatment of PMNE has traditionally been associ-
ated with a large S-shaped incision to release all potentialsites of compression, but carries with it a great disadvantage
in the extensive scar tissue that is created.
Our technique has consistently focused on being mini-
mally invasive, releasing the most prominent, and consis-
tently present, constricting structure at the elbow —the
lacertus fibrosus. The advent of wide-awake surgery, imply-ing use of local lidocaine –epinephrine infiltration and no
tourniquet, has allowed the opportunity to treat these
patients with a minor surgical procedure where the returnof strength in the median innervated muscles can be appre-
ciated perioperatively.
Anesthesia
The patient is anesthetized 30 min preoperatively using 20 –
30 ml 1 % lidocaine (10 mg/ml) with epinephrine (5 μg/ml)
with 2 –3 ml sodium bicarbonate (50 mg/ml) solution added,
respectively. Using a 27-in.-gauge needle, the anesthesia is
slowly infiltrated from the medial elbow crease and oblique-
ly 4 cm distally and centrally to cover the course of the
lacertus fibrosus (Fig. 1). Immediately preoperatively, and
after infiltration of the local anesthesia, weakness in FPLand FDP II is again assessed to confirm the diagnosis and
rule out the pain block of the anesthetic as a cause of
returned muscle strength.
Fig. 1 Preoperative photo illustrating the area where the infiltration
anesthesia was applied to a patient planned for lacertus tunnel release.Thirty milliliters of 1 % lidocaine with epinephrine was administered
subcutaneously 30 min preoperatively. The dotted lines mark the area
of subcutaneous infiltration, covering the area of the lacertus fibrosusand the medial/central aspect of the elbow and forearm. Skin pallor canbe seen as a result of the vasoconstrictive effect from the epinephrine.
BT=biceps tendon; ME=medial epicondyle42 HAND (2013) 8:41 –46 | 515a2f826486c30c068d75b1dc5c0dd1d8277373 | page_0001 |
Surgery (Video 2)
The surgery is performed with the patient awake and no
tourniquet. A 2 –3-cm transverse incision is placed in the
flexion crease of the cubital fossa, from 1 cm medial of the
biceps tendon to 2 cm lateral of the medial epicondyle (seeFig. 2a). Careful dissection is made subcutaneously to the
pronator teres fascia (Fig. 2b), taking great care to identify
and protect branches of the medial antebrachial cutaneousnerve. The pronator teres fa scia is incised and followed
laterally, allowing exposure of the lacertus fibrosus
(Fig. 2c), which is subsequently divided. By retracting the
pronator teres muscle medially, the median nerve is readily
exposed (Fig. 3d). Any focal adherences to the underlying
brachialis muscle may then be released. At this point, thestrength of the FPL and FDP II is again tested intraopera-
tively before the skin is closed, as return of muscle strength
is usually immediate after proper release of the nerve (seeVideo 2). After cauterization, the wound is closed with
interrupted sutures, a small soft dressing applied and imme-
diate mobilization encouraged. A load of 1 kg is alloweduntil suture removal 2 weeks postoperatively. Full load is
permitted 4 weeks postoperatively. Patients with no manual
labor return to work within 1 –2 days postoperatively.
Results
Prospective Follow-Up
Our results of a 5-year retrospective follow-up of 82 patients
with PMNE released through surgical decompression has
previously been reported [ 10]. To enhance the objective find-
ings in these patients, we have followed our patients prospec-tively with quick DASH, including work and activity-
related DASH scores, preoperatively and 6 months post-
operatively. In addition, the patients answered postoper-ative questions related to their perceived subjective
functions, as recorded using visual analog scales (VAS).During 2011, 44 patients with PMNE were surgically
released and followed prospectively, 22 women/22 men,
mean age 48.8 (range 25 –66). The patients were equally
distributed between right/left hands (23/21), and the domi-
nant hand was treated in 56 % of cases.
The average preoperative quick DASH was 35.4 (range
6.8–77.2); work DASH, 44.3 (6.25 –100); and activity
DASH, 61.6 (12.5 –100). 71.1 % completed the 6-month
follow-up, and the average postoperative quick DASH was12.7 (range 0 –43.1), which is a statistically significant re-
duction ( p<0.0001; Student ’s paired t test). Similarly, the
work and activity DASH was significantly reduced(p<0.001) to 12.5 (0 –75) and 6.25 (0 –50), respectively.
At the 6-month follow-up, the patients were asked three
questions: (1) persistent numbness in the operated hand?(V AS 0=no paresthesias, 10=severe paresthesias); (2) persis-
tent pain in the operated elbow/hand? (V AS 0=no pain, 10=
extreme pain); and (3) satisfaction with surgery? (V AS 0=completely dissatisfied, 10=entirely satisfied). The average 6-
month V AS scores were as follows: (1) numbness, V AS 1.3;
(2) pain, V AS 0.9; (3) satisfaction, V AS 8.8 (Table 1).
Discussion
In this paper, a clinical triad to diagnose PMNE at the level
of the lacertus fibrosus is described: weakness in muscles
innervated distal to the lacertus fibrosus (FCR, FPL, andFDP II); pain over point of compression (lacertus level); and
positive scratch collapse tes t. The prospective analysis
shows a statistically significant postoperative improve-ment in DASH scores (quick, work, and activity DASH,
p<0.001) as compared to preoperative measurements, as
well as a high subjective satisfaction (VAS) with theresult of surgery.
Pronator syndrome has traditionally been described as an
elusive diagnosis, where all seven possible points of nervecompression from the mid-upper arm to the mid-forearm,
should be released through a large S-shaped incision [ 14].
Fig. 2 Surgical release of the median nerve at the level of the
lacertus fibrosus (lacertus tunnel syndrome). aA transverse inci-
sion is placed from just medial to the biceps tendon ( BT)t oj u s t
lateral to the medial epicondyle ( ME). Following blunt dissection
of the subcutaneous tissue, the p ronator teres fascia is opened,and the muscle was retracted medially. bThe lacertus fibrosus
(LF) is carefully isolated ( arrow ) and divided in its entire length
while the median nerve is kept under visual control. cWith the
pronator teres retracted medially, the median nerve ( MN) is easily
seen after division of the LFHAND (2013) 8:41 –46 43 | 515a2f826486c30c068d75b1dc5c0dd1d8277373 | page_0002 |
Fig. 3 a Illustrations of a transverse section of the “lacertus
tunnel ”at the volar and medial aspect of the elbow, modified
from Grant ’s Atlas of Anatomy(2). The medial humeral trochlea
(MHT ) constitutes the bottom of the tunnel; the lateral and medialwalls are the brachialis ( BR) and pronator teres ( PT) muscles,
respectively, and the roo f is the lacertus fibrosus ( LF).MN=
median nerve; A=brachial artery; BT=biceps tendon. bzoomed
view of the lacertus tunnel
Table 1 Summary of average pre-/postoperative DASH scores and
subjective visual analogue scales (VAS) at 6-month follow-up. The toptable (blue) illustrates the preoperative and 6 months postoperativequick DASH (q-DASH), work DASH (w-DASH), and activity DASH
(a-DASH), following proximal median nerve release at the level of thelacertus fibrosus. *=significant difference ( p<0.001). The bottom table
(red) illustrates the results of subjective visual analog scores (VAS)regarding persistent numbness (0=no paresthesias, 10=severe pares-thesias); persistent pain (0=no pain, 10=extreme pain); and satisfac-
tion with surgery (0=completely dissatisfied, 10=entirely satisfied)
q-DASH pre
q-DASH post
w-DASH pre
w-DASH post
a-DASH pre
a-DASH post
0 1 02 03 04 05 06 07 0DASH scores
Numbness
Pain
Satisfaction
0123456789Subjective Scores (VAS)*
*
*44 HAND (2013) 8:41 –46 | 515a2f826486c30c068d75b1dc5c0dd1d8277373 | page_0003 |
This surgical technique is technically demanding and, most
importantly, associated with a high morbidity as it leaves the
patient with severe scarring in the arm.
Two recent publications advocating minimally invasive
treatment of pronator syndrome, one open technique [ 23]
and one arthroscopic [ 13], have challenged the idea of a large
incision to treat pronator syndrome. The open technique, as
published by Zancolli et al. [ 23], describe a syndrome where
the patient has concomitant carpal tunnel syndrome, weaknessin the superficialis muscles (FDS IV) and paresthesia elicited
upon compression of the nerve at the level of the superficialis
arcade. Both techniques have in common that they primarilyrelease the “fibrous arcade ”of the superficialis muscles, lo-
cated approximately 7 cm distal to the elbow crease. The
clinical findings and surgical release thus differ greatly fromthat described in this manuscript.
In 1945, Sunderland described the internal topography of
the radial, median, and ulnar nerves from the axilla to thehand [ 21]. Based on this original investigation, he conclud-
ed:“The nature, extent and peripheral effects of injury
sustained by a nerve as the result of trauma are influencedby the funicular pattern at the site of injury. ”In an early
stage of nerve compression syndrome, there is no actual
injury to the nerve, rather ischemic changes and alterations
in axonal transport, but the correlation to funicular patterns
is still relevant.
Akin to the carpal tunnel, at the level of the lacertus
fibrosus, the median nerve passes through a “tunnel, ”where
the bottom, the lateral wall, and medial wall are the medialtrochlea of the humerus, the brachialis, and the pronator
muscles, respectively, and the roof is the lacertus fibrosus
(Fig. 3). According to Sunderland [ 21], at this “lacertus tun-
nel,”the median nerve is organized such that the branches to
the pronator teres and FCR are located anteriorly, and the
branches to the FPL and FDP II, medially. At the level ofthe superficialis arcade, on the other hand, the anterior and
superficial branches correspond to the FDS and the cutaneous
innervation of the hand. These anatomical variations are mir-rored in the clinical differences and varying levels of surgical
release in the Zancolli publication and the present manuscript,
and may be understood as two independent entrapments of themedian nerve proximal to the carpal tunnel —one at the level
of the superficialis arcade, the other at the level of the lacertus
fibrosus. A more distinct and accurate description would thusbe to name these the “superficialis syndrome ”and“lacertus
tunnel syndrome ”(LTS), respectively.
The scratch collapse test (SCT) has previously been shown
to be a sensitive clinical provocative maneuver in diagnosing
peripheral nerve entrapments, with a higher sensitivity than
traditional clinical tests, such as Tinel ’s and compression tests,
in diagnosing carpal tunnel and cubital tunnel syndromes [ 6],
as well as peroneal nerve entrapment [ 9]. The use of the SCT
has also been suggested as an important tool in differentiatingbetween levels of ulnar nerve entrapment at the level of the
elbow [ 4]. Similarly, SCT was an important adjunct in diag-
nosing LTS, along with specific muscle weakness and pain atthe level of entrapment (lacertus tunnel).
In conclusion, proximal median nerve entrapment at the level
of the lacertus fibrosus should be referred to as lacertus tunnelsyndrome, LTS, to distinguish it from other levels of median
nerve entrapment. It is a clinical diagnosis based on three
distinct clinical findings: weakness, pain over point of compres-sion, and positive scratch collapse test. Surgical release in local
anesthesia allows for a safe, ambulatory, and cost-efficient
procedure with low morbidity. Larger, multi-center, and pro-spective studies are planned to further enhance our understand-
ing of this prevalent, but frequently misunderstood, diagnosis.
Conflict of Interest The authors declare that they have no conflict of
interest.
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Synthetic biology to access and expand nature’s chemical
diversity
Michael J. Smanski1, Hui Zhou2, Jan Claesen3, Ben Shen4, Michael Fischbach3, and
Christopher A. Voigt2
1Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute,
University of Minnesota – Twin Cities, Saint Paul, MN, 55108, USA
2Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
3Department of Bioengineering and Therapeutic Sciences, University of California-San Francisco,
San Francisco, CA 94158
4Departments of Chemistry and Molecular Therapeutics, The Scripps Research Institute, Jupiter,
FL 33458
Abstract
Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex
molecules with diverse applications, from medicine to agriculture and materials. Economically
accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug
discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous
undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases
of DNA, are controlled by complex regulatory networks, and the most interesting molecules are
made by non-model organisms. Advances in synthetic biology address these issues, including
DNA construction technologies, genetic parts for precision expression control, synthetic regulatory
circuits, computer aided design, and multiplexed genome engineering. Collectively, these
technologies are moving towards an era when chemicals can be accessed en mass based on
sequence information alone. This will enable the harnessing of metagenomic data and massive
strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design
pathways to complex chemicals not found in nature.
Introduction
Natural products (NPs) are specialized metabolites produced by plants, animals, and
microorganisms with diverse chemical structures and biological activities. These molecules
are valuable in the clinical setting, with half of small molecule drugs approved during the
past three decades being derived from NPs1. While NPs are prevalent in the treatment of
infection2, cancer3, and as immunosuppressive agents4, they have also made it into
commercial products as antivirals, anthelmintics, enzyme inhibitors, nutraceuticals,
Correspondence and requests for materials should be addressed to C.A.V . ([email protected]).
HHS Public Access
Author manuscript
Nat Rev Microbiol . Author manuscript; available in PMC 2016 October 04.
Published in final edited form as:
Nat Rev Microbiol . 2016 March ; 14(3): 135–149. doi:10.1038/nrmicro.2015.24.
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polymers, surfactants, bioherbicides, and vaccines5. In many NP-producing organisms, all of
the genes required for regulation, biosynthesis, export, and self-resistance are co-localized in
the genome in compact ‘biosynthetic gene clusters’ (BGCs) (Figure 1a).
Claims that natural products are an inexhaustible resource6 are based on the disparity
between the staggering biological and chemical diversity on the planet and the relatively
low-throughput methods currently available to characterize these compounds. In the coming
decades, advances in technology will close this gap and allow for a more systematic
characterization of global NP production. Improving bioinformatic methods, combined with
the dramatic rise in sequenced genomes, is shedding light on the potential number of
undiscovered natural products (Figure 1b)7–17. In Streptomyces alone, conservative
estimates put the number of natural products at 150,000, of which <5% have been
discovered18. Bioinformatic investigations of hundreds of genomes across genera estimate
that there are 100,000s of NPs19, and the inclusion of less-studied classes, such as
saccharides and lipids, substantially add to the number of molecules12.
Currently, our ability to mine bacterial genomes to produce NPs is unable to keep pace with
the identification of new BGCs by DNA sequencing and bioinformatics. However, the cost
of DNA sequencing and synthesis continues to drop (Figure 1c) and future advances are
projected to quickly make it possible to build the DNA for many pathways. This leaves
design as the biggest remaining issue, where it is still challenging to rationally compose a
DNA sequence for a large pathway that will be functional in a model production host.
Engineering NP biosynthesis is still difficult for several reasons. Factors like transcription,
translation, protein-protein interactions, cofactor and precursor availability, export, and self-
resistance all need to be accounted for in a final production strain. In addition, many of the
organisms that harbor these BGCs are difficult to manipulate or cultivate20,21 and the
transfer of a BCG to a new host, for which there are successful examples22, is by no means
trivial.
The goal of this review is to highlight emerging technologies relevant to engineering multi-
gene systems with a special focus on the application of methods from synthetic biology to
the engineering of biosynthetic pathways and NP-producing organisms. Many of these
technologies were developed in model lab organisms like E. coli or S. cerevisiae , so when
relevant, technical difficulties associated with moving these into major NP production strains
are discussed. Other areas relevant to NP discovery, including methods of NP identification,
manipulation of global and pathway-specific regulators, and prioritization of BGCs
identified via genome sequencing efforts will not be discussed in detail, as these were
reviewed recently23.
Synthetic biology to accelerate natural product discovery and production
Engineering NP biosynthesis draws tools from a variety of subfields in genetic engineering
and chemistry. For example, protein engineering seeks to modify the properties of individual
proteins, including the activity/specificity/stability of enzymes and in the recombination of
domains to diversify the products of large PKS/NRPS “assembly lines”24. Metabolic
engineering is focused on understanding how multiple enzymes assemble into a pathway and Smanski et al. Page 2
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how this impacts metabolic fluxes in the larger natural network25. For example, the flux of a
precursor to a natural product could be boosted as part of optimizing the titer. Applied
microbiology and strain engineering have been critical for identifying high-producing
organisms and optimizing the titer and performance in a fermentation through processes
such as the random chemical mutagenesis of the genome26. Synthetic biology has focused
on tools to accelerate and increase the scale of genetic engineering27,28. The scope of this
review is to cover recent advances in synthetic biology in the light of how they will impact
the field of NPs, including the following technologies:
• Abstraction of genetic functions into “parts.” There has been an emphasis
on creating genetic parts, such as promoters, that generate precise levels of
gene expression29–32. There has been focus on generating large libraries of
well-characterized parts and the development of biophysical/
bioinformatics models to predict part behavior33–36. Part libraries for
different organisms will aid the transfer of BGCs between hosts37–41.
• Large-scale construction technologies . DNA synthesis capacity has
exploded over the last decade and it is routine to synthesize the
20kb-100kb needed for a large gene cluster42–44. In addition, new DNA
assembly methods enable the rapid construction of different part
permutations or to substitute many parts in a single step45–47.
• Design automation . New computer aided design methods and work
environments accelerate the process of designing a genetic system,
scanning the system for errors, and to analyze screening and –omics
datasets48–50.
• Synthetic regulation . Genetic circuits have been constructed that function
as logic, timers, switches, and oscillators51–53. Sensors have also been
developed that respond to many inducible inputs as well as metabolite
levels. These could be incorporated into natural product pathways to
control the timing of expression of different genes or to implement
feedback in response to a toxic intermediate54.
• Genome editing for host design . It is often desirable to make many
simultaneous genomic changes. Methods, such as CRISPR-Cas9, can
target essentially any region of the genome and have been shown to
function in many species, including several host species well-suited for
industrial production of small molecules55–58.
Reducing genetics to genetic parts
Natural product BGCs are large and unwieldy59. They can comprise several dozen genes,
arranged in one or many operons facing either direction. Their expression often relies on
regulatory elements that are overlapping or imbedded in neighboring genes. They are under
multiple layers of complex regulation including transcriptional and translational control.
These factors makes engineering gene expression in BGCs technically difficult. This
organization is in contrast to the concept of ‘genetic parts,’ which are units of DNA with Smanski et al. Page 3
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defined and modular function that replace native regulation to provide finer control over
biological processes60.
Applying a parts-level approach to multi-gene systems is facilitated by ‘refactoring’, in
which the natural genetics are re-written to make the systems more amenable to engineering
efforts (Figure 2a)43,61. A refactored gene cluster has several advantages that lend
themselves to high-throughput applications. First, the process of refactoring severs the native
regulation, which is usually incompletely understood, and allows for synthetic control of
gene expression. Second, it leads to a modular genetic architecture, which facilitates part-
swapping and combinatorial optimization. An example of an application would be in the
creation of diverse compounds by substituting variations of enzymes from homologous
clusters (Figure 2b). Further, the expression levels of the enzymes are important and it is
more difficult to control if the regulatory parts ( e.g., RBSs) cannot be exchanged without
leading to a web of other effects. Refactored BGCs can undergo wholesale swapping of
genetic parts to optimize expression levels and increase titers. The concept of refactoring has
started to be applied to BCGs62,63 (Figure 2c) around the substitution of some synthetic
regulatory elements and as the parts and tools improve, this will expand to include the
complete elimination of native regulation.
Precision control of gene expression
Many metabolic pathways and BGCs are highly sensitive to gene expression, where small
changes can cause a loss of activity64,65. Recent work to create large libraries of regulatory
parts enable the graded control of gene expression over many orders of
magnitude30,33,37,66–68 (Figure 3a). In addition, computational methods have been
developed that will design a new part based on biophysical models of transcription or
translation34,35,69,70. While much work has been done in E. coli , there have been efforts to
expand part libraries into other relevant organisms, including Streptomyces37–39, Bacillus40,
and fungi41,66,71(Figure 3b). This can often be stymied by issues that are taken for granted in
model organisms. For example, single cell techniques, such as flow cytometry and
fluorescent reporters, are difficult to use in Streptomyces because they have multicellular
branched growth patterns72,73, differences in the DNA copy number, and the stochastic
nature of gene expression seen during mycelial growth72. Different paradigms for measuring
part function are needed for these hosts. Part design is also complicated by other issues; for
example, the high GC content of some genomes make it difficult to codon optimize genes
and design RNA parts, including RBSs36,74.
In synthetic biology, the concept of the “expression cassette” has been expanded to include
insulators that increase the reliability of part function in different genetic
contexts29–32(Figure 3a). This arose out of observations that different combinations of parts,
for example promoters and RBSs, can lead to unexpected behaviors30,75. Some examples
include ribozymes that decouple the promoter from the 5 ′-UTR76 and bi-cistrons that
decouple the 5 ′-UTR from the RBS32. These allow the promoters and RBS to be swapped to
vary expression levels without impacting the behavior of neighboring parts. Similarly, long
promoters and strong terminators have been developed to transcriptionally insulate the
genes33,77. Collectively, this has led to genetic architectures that are more focused on the Smanski et al. Page 4
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control of individual genes as individual cistrons, as opposed to their organization into
operons. Adopting these design principles for BGCs will be important for combinatorial
optimization or in the exchange of parts to create chemical diversity.
High-throughput genetic optimization of multi-gene systems
Accessing new NPs from genome sequence information requires that the BGCs contained in
sequence databases be converted into physical DNA constructs. High-throughput fabrication
allows many designs to be tested in parallel, thereby increasing the probability of identifying
a functional construct. This is beneficial both for chemical diversification via combinatorial
biosynthesis78–81, as well as for genetic optimization of pathway performance662,64.
There are two DNA construction technologies relevant to BGCs (Figure 4a). The first is de
novo synthesis where genes or entire clusters are chemically constructed, typically by
synthesis companies82. The cost has dropped dramatically in the last decade and it is
possible to order hundreds of individual genes or full clusters43,63,83. While the cost has
declined significantly, it is still expensive to build large clusters and building comprehensive
sets of clusters out of the sequence databases is prohibitive. However, for NP biosynthetic
classes such as RiPPs, this low cost synthetic DNA can be leveraged for combinatorial
generation of new derivatives84–86.
The second is DNA assembly, which constitutes the combination of parts to build a larger
construct46,87–94. This enables many variants of gene clusters to be built based on a set of re-
used underlying parts45. This is significantly cheaper that constructing de novo clusters for
each variant that one wants to test. Many assembly methods are now available, including
Isothermal assembly91, Golden Gate assembly95, ligase cycling reactions94, scarless
stitching45, and recombination-based methods92,96. Automating these techniques using
liquid handling robots enables hundreds or thousands of permuted combinations to be
built45,48,79,80,85,97.
Combinatorial optimization will be important in optimizing a BCG and transferring it
between hosts. Epistasis in the expression levels of biosynthetic genes points to the
importance of combinatorial optimization methods that look at more than one variable at a
time64,98. Once pathways grow beyond a small handful of genes, complete exploration of
the combinatorial gene expression space is impractical or impossible. This combinatorial
space can be reduced via Multivariate Modular Metabolic Engineering by using a priori
knowledge of a biochemical pathway ( e.g., enzyme kinetics, order of reactions, or pathway
branching architecture) to constrain groups of genes into a small number of operons99.
Combining metabolic modeling and RBS design, production levels of the isoprenoid
neurosporene could be tuned over a continuous range from 0-300 μg per gram of dry cell
weight36. Another approach is to use Design of Experiments (DOE) to reduce the number of
experiments required to search a combinatorial space. These require that variables effecting
construct performance can be readily manipulated, but do not require an understanding of
the underlying biology. Recent examples in the neurosporene and violacein biosynthetic
pathways shows that following a fractional factorial experiment, new positions in the
sequence space can be accurately predicted36,71.Smanski et al. Page 5
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Combinatorial assembly has been applied to the optimization of several NP pathways, as
well as in the creation of chemical diversity. Several examples of optimizing BGCs,
including for heterologous production of artimisinin65, taxadiene64, and opiate alkaloids100
point to the importance of combinatorial engineering, as optimal production levels do not
result from a blanket gene over-expression. Using a Gibson assembly protocol optimized for
high GC content, the pathway specific regulators of pristinamycin biosynthesis were
mutated in a combinatorial fashion to increase production levels to over 1 g/L101. An
improved three-gene pathway for catechin production was created via combinatorial
assembly by drawing from eight homologous biosynthetic genes from different plant
species102. Certain classes of NPs, for example indolocarbazoles, have been greatly
expanded using combinatorial DNA assembly with more than fifty derivatives created to
date78,103. Finally, combinatorial assembly has been applied to probe the desire rules
underlying large multi-modular enzymes79–81,85,104,105. For example, promiscuous
polyketide donor or accepter modules were identified79.
Host Transfer
Transferring a BGC between hosts is important for NP discovery, diversity screening, and
optimization. This is particularly true if the BCG only appears in a sequence database and its
native organism is unknown or inaccessible. Transferring a BCG would enable the new host
to make the encoded compound.
However, the direct transfer of a BCG between even similar species can result in dramatic
changes in the timing and relative expression levels for pathway genes (Figures 4c)106. This
is unsurprising given the performance of genetic parts depends on components of the host
cell’s machinery75. These host-context effects can result from unintended crosstalk or
interactions with native regulatory proteins107, from limitations in host resources available
for expressing heterologous constructs108, and for NP production, from crosstalk with
endogenous biosynthetic pathways. For example, transfer of the platencin gene cluster from
S. platensis to the model host S. lividans resulted in the excess accumulation of shunt
metabolites (Figure 4c,d)106. This was correlated with substantial changes in gene
expression patterns. A likely explanation is that improperly balanced expression levels in the
heterologous host led to build-up of pathway intermediates, which were then subject to
modification by endogenous biosynthetic enzymes and thus diverted away from the desired
product (Figure 4d). Even moving multi-gene systems just between different strains of the
same species can likewise negatively impact performance107,109,110.
One way to transfer the BCG more effectively would be to exchange parts for those that are
known to function in the new species (Figure 4e). Effort could be taken to ensure that the
desired expression levels are reached in the new host chassis. For example, differences in the
anti-Shine-Dalgarno sequence in the small ribosomal subunit mean that RBS strengths are
effected upon transfer to disparate hosts and this requires that all the RBSs be
simultaneously redesigned36. This type of wholesale reassignment of regulatory parts is
much easier in refactored genetic systems, whose modular architectures allow them to be
built up from parts quickly. Also important is increasing the size of characterized part
libraries for NP-relevant species37–41,66,67,71,90,111,112. For complex multigene systems, the Smanski et al. Page 6
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dramatic design changes that accompany host transfer will likely require further genetic
optimization via combinatorial DNA assembly to re-tune expression levels and improve
performance (previous section)45.
Genetically-encoded biosensors for biosynthetic pathway engineering
The design and construction of DNA libraries has accelerated to the point where screening
and making sense of this diversity is the bottleneck. While bioassay-guided prescreens or
selections can reduce the number of strains that need to be investigated in detail26, these do
not replace direct measurements of titer. Current analytical chemistry methods have reduced
analysis time to <3 min per sample113, but this still limits throughput to ~103 per day. As an
alternative to analytical chemistry, in vivo biosensors translate information about a chemical
signal, i.e. the concentration of a natural product, into light or fluorescence-based output that
can be measured by flow-cytometry to screen thousands genotypes per second114. As an
example of the power of this approach, the inherent fluorescence of the carotenoid
astaxanthine was exploited to track titers in single cells by flow cytometry and cell-sorting,
enabling a 10,000-fold enrichment of over-producing strains compared to plate-based
techniques115.
Intracellular biosensors can be broadly grouped into three categories, RNA-based, protein-
based, or enzyme-based, according to their biomolecular make-up and mechanism. RNA
aptamers that bind to small molecule signals to actuate a response have been linked to
readouts including fluorescence, enzyme activity, cell mobility, or viability114,116. There are
diverse strategies for designing RNA biosensors117–125. RNA aptamers have been used to
build biosensors for natural products and intermediates, including theophylline121,
tetracycline124, neomycin126, tobramycin127, dopamine128, and ochratoxin A129.
Protein biosensors function by transmitting molecular binding information into a measurable
output, usually in the form of allosterically-regulated transcriptional activators or repressors
controlling fluorescent protein expression. Naturally-evolved biosensors ( e.g. Figure 5a,b)
can detect a wide range of molecular scaffolds, including tetracyclines130,131, cationic lipids
and plant alkaloids132, and anthraquinones133. There are >4000 TetR proteins identified in
sequence databases, and only a small fraction of the ligands are known134. While protein
biosensors have been found or engineered for a number of target molecules, including
aromatics135,136 and branched-chain amino acids 137, these will need to be repurposed to
sense new molecules for generalized use in NP discovery and optimization pipelines. Cirino
and co-workers engineered the transcriptional activator AraC to recognize either mevalonate
(a key precursor for isoprenoids)138 or triacetic acid lactone (a simple polyketide
product)139. In an alternative to modulating gene expression, the Liu group has engineered
protein biosensors that sense small molecule ligands to actuate an intein-splicing
event140,141, thereby allowing a diverse set of proteins or enzymes to be activated only in the
presence of the ligand.
Enzymatic biosensors recognize the desired metabolite and convert it into a pigmented or
fluorescent molecule that can be easily detected by spectrophotometry or flow cytometry. To
optimize the production of L-DOPA in yeast, Dueber and co-workers expressed a DOPA Smanski et al. Page 7
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dioxygenase intracellularly142. The DOPA dioxygenase results in an extradiol ring cleavage
of L-DOPA to produce the aldehyde-containing betalamic acid, which spontaneously reacts
with cellular amino acids to form fluorescent imines known collectively as betaxanthins143.
Whole cell biosensors can be created without the need for complicated protein or aptamer
engineering144. For example, recombinant E. coli made auxotrophic for mevalonate shows
concentration-dependent growth rate changes in the presence of extracellular mevalonate145.
By expressing GFP within this strain, mevalonate levels can be detected in high-throughput
from the culture broths of a production strain145. In principle, this strategy can be used to
quickly engineer a whole cell biosensor to any molecule that can be made essential for strain
viability146.
Potential applications for synthetic genetic circuits
The temporal control of expression is often important in building complex chemicals and
materials147,148. In natural systems, this is implemented via regulatory networks consisting
of interacting proteins, RNA, and DNA that collectively work to perform a computational
operation. Synthetic genetic circuits have been built where a target behavior is achieved by
artificially connecting regulatory proteins (Figure 5). This has been used to build cascades,
bistable switches, pulse generators, oscillators, feedback/feedforward motifs, and logic
gates29,51. NP pathways reveal complex and intricate control mechanisms including many of
these same behaviors149–151. However, the naturally evolved regulation is not required for
high-level production in defined culture conditions. In fact, its disruption often leads to
improved production152–154. Replacing native regulation with synthetic circuits may
implement the necessary feedback and dynamics without also having the environmental
control that can inhibit production.
Feedback and feedforward regulation has been used to tie the accumulation of early-stage
pathway intermediates with the expression of downstream processing genes. Feedforward
regulation is seen in many NP biosynthetic pathways. In actinorhodin biosynthesis, the
accumulation of pathway intermediates triggers expression of the efflux pump to export the
final product133. In this case, the feed-forward motif helps to prevent the cell from
deleterious effects of the accumulating high concentrations of the antibiotic within the cell.
Modeling of a synthetic pathway for para-aminostyrene biosynthesis suggests that higher
titers can be attained with dynamic regulation incorporating feedback/feedforward
regulation compared to static regulation (Figure 5c)155. Using a fatty-acid biosensor to add
feedforward/feedback regulation into a synthetic fatty acid ester pathway allowed Zhang et
al. to boost bio-diesel production to 28% theoretical yield (Figure 5d)54. Additionally,
positive and negative feedback loops can be exploited to control the allocation of cellular
resources to secondary metabolism (Figure 5e)156.
Synthetic circuits that act as metabolic “control valves” have been used to redirect carbon
flux from primary to secondary pathways157,158. This dynamic control over central carbon
metabolism is important, because if this diversion is constitutive, it slows growth to the point
of decreasing productivity. Similar dynamic switching is seen in Streptomyces prior to
antibiotic production159. The expression of housekeeping genes, particularly those involved Smanski et al. Page 8
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in translation, is strongly diminished prior to entering stationary phase, at which time
actinorhodin and undecylprodigiosin production begins.
Control over the timing of gene expression can also be achieved using synthetic bistable
switches, which reset slowly over time (Figure 5f)160. Bistable switches appear in many NP
producing organisms, including Streptomyces coelicolor where it controls cryptic polyketide
(cpk) production151. Engineered timing circuits can be used to separate cell growth and NP
production phases, particularly in strains for which the regulatory connections to the natural
metabolic switch159 have been severed.
Genetic logic gates allow multiple input signals to be integrated before a pathway turns on.
There is some evidence of logic in natural NP pathways, for example, the actinorhodin gene
cluster is controlled by metabolite concentrations, stress response, and development
program149,150. This can be used to turn on different sets of genes under different
environmental conditions, the specificity of which improves as more signals are integrated.
In synthetic biology, many logic gates have been built29,52,107,110,161–163. Connecting
synthetic multi-input circuits to NP pathways could allow cells to sense cofactor levels,
precursor abundance, dissolved oxygen content, or ATP charge before deciding whether or
not to commit to NP biosynthesis. Such intracellular checkpoints could prevent the
accumulation of unwanted intermediates and byproducts by cells that are not capable of
making the final product.
CRISPRi has emerged as a powerful method to regulate gene expression, including those in
the genome, for both prokaryotes and eukaryotes. It is based on the expression of a
catalytically-inactive dCas9, which can be directed to a target when a sgRNA is
transcribed164. dCas9 can serve as either a repressor or activator (CRISPRa) depending on
domains to which it is fused165. CRISPRi has been shown to work in E. coli164, fungi165,
actinobacteria55, and plants166. Other organisms relevant to NP biosynthesis, such as
cyanobacteria167, burkholderia168, pseudomonads169, and myxobacteria170, have
endogenous CRISPR systems but have not as of yet been exploited as hosts for CRISPRi
regulation. CRISPRi has already been exploited to control metabolic fluxes via multiplexed
gene repression of endogenous pathways in E. coli171 and in heterologous pathways in yeast
(Figure 5g)172.
New tools for combinatorial genome-scale engineering
Mutations in the genome outside of the BGC are required to optimize the titers of a NP.
Some industrial strains can achieve gram per liter quantities, and this is usually achieved via
random mutagenesis of the genome and screening26. Originally, the genetic diversity was
generated using techniques such as chemical mutagenesis, but this has gotten more
sophisticated with improved molecular biology methods. For example, the whole-genome
shuffling of a tylosin producer yielded the same 6-fold improvement in 24,000 assays that
had taken Eli Lilly 20 years and 1 million assays to achieve by classical methods173.
Synthetic biology offers new techniques for generating genome diversity, from methods to
replace parts or make defined mutations in a multiplexed manner to genome construction via
de novo synthesis44,174–176.Smanski et al. Page 9
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Multiplexed genome engineering strategies offer the ability to precisely target hundreds of
loci in the genome for over- or under-expression in parallel. The first demonstration of
massively-multiplexed recombineering in E. coli exploited oligo-mediated allelic
replacement177. By mimicking Okazaki fragments at the replication fork, exogenous
ssDNAs are able to anneal with the lagging strand of the genome serving as primers for
DNA elongation and then get incorporated, which leads to simultaneous directed
mutagenesis at multiple sites in the genome178. MAGE (Multiplexed Automated Genome
Engineering) automates this process, which allows combinatorial exploration of mutations in
a continuously evolving population. For example, twenty endogenous E. coli genes were
targeted to optimize lycopene production and billions of variants were screened177. MAGE
has also been used to insert regulatory parts into the genome, such as N- and C-terminal
tags179 and T7 RNAP promoters176,180. Recombineering has been shown to work in diverse
organisms, including lactic acid bacteria181, mycobacteria182, corynebacteria183, and
fungi184. Getting MAGE to work in Streptomyces will be challenging due to the high GC-
content, the lack of characterization of mismatch repair in this species, and the fact that the
expression of some genes required by the technique (e.g., bet) is not likely to produce
functional proteins183, and transformation is significantly more challenging185. MAGE has
already been shown to work in yeast and can be applied to pathways transferred to this
host184 and it may work in other NP-relevant fungi.
TRMR (trackable multiplex recombineering) is a related method, which was developed to
rapidly map the effects of more than 95% of E. coli genes onto specific traits174. TRMR
exploits array-based DNA synthesis to create barcoded oligonucleotides that target over
4,000 genes for either over-expression or repression. Following phenotypic enrichment,
deep-sequencing allows the targeted mutations to be quickly mapped to identify the causal
mutations, generating massive amounts of sequence-to-phenotype relationships174,186.
Combining TRMR with multiplexed recombineering allowed the identification of 27
genome modification targets that accelerated growth in a target medium186.
CRISPR techniques have revolutionized multiplexed genome engineering55–58,187–197. The
Cas9 nuclease can be targeted to specific sequences by transcribing a sgRNA (Figure 6a).
This system has been shown to work in nearly every organism that has been tried, including
prokaryotes 55,57,171,187,189–191,195 such as Streptomyces (Figure 6b), eukaryotes58,188,198,
and higher organisms of relevance to NP production56,199. The lack of a canonical non-
homologous end joining (NHEJ) DNA repair in some prokaryotes, including Streptomyces ,
lowers the efficiency of gene inactivation when CRISPR-Cas9 is used alone55,200. However,
the efficiency of gene inactivation can be increased to over 75% by (i) including a double-
stranded ‘repair fragment’ that can close the double-stranded DNA break via homologous
recombination57, or (ii) reconstituting a NHEJ pathway through heterologous expression of
the ligase LigD during genome editing55. This system can be used to make five mutations in
a single step (Figure 6c)188, knockout 31 kilobase gene clusters57, and insert DNA up to 9
kilobases in length198.Smanski et al. Page 10
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Conclusions
New strategies from synthetic biology are enabling the engineering of large systems
comprising many genetic parts, the control of gene expression with synthetic regulation, and
efficient genome editing. New tools exist that provide precise control of gene expression
from synthetic constructs, and the fabrication of large systems is made easier by abstracting
designs in a parts-based approach to genetic engineering. While many of the approaches in
this review were developed in a model organism, such as E. coli , over the last few years they
have been increasingly ported to organisms of more direct relevance to NP discovery and
production.
For NP chemists and biologists, the challenge will be determining how best to leverage the
latest technologies in DNA fabrication and genetic control to probe NP pathways in new and
insightful ways. Much in the same way that recombinant DNA technology revolutionized
our ability to approach the molecular details of biology from a reductionist point of view, the
ability to rapidly build large libraries of specifically designed gene clusters will provide
greater opportunity to explore the effect of genetic design on the functional expression of
BGCs. Continued research into the detailed regulatory mechanisms of employed within
natural BGCs and the biochemistry of NP biosynthetic pathways will be paramount for
forming hypotheses that can be tested using new bottom-up techniques.
Current high-throughput and multiplexed genetic engineering strategies can be harnessed to
develop applications for NP-producers outside of the fermenter as well (Figure 7). This
could have applications in environmental sensing, for example producing a small volatile
metabolite in response to metal contamination in soils, or in the production of therapeutics
by probiotic strains, for example making antibacterial compounds in response to a pathogen
in the GI tract201–204. NPs are already used extensively in agriculture for crop protection205,
and gaining fine-tuned control over production dynamics either in soil microbial
communities or in crop plants themselves could impact food production.
This is an exciting time for NP biosynthesis. The number of possible applications for NPs in
medicine, industry, and agriculture is vast. The recent explosion in DNA sequencing
technologies has revealed that BGCs for NP production are more widespread than
previously imagined12,19,206, and the ability to ‘write’ DNA into synthetic constructs is now
catching up. New approaches for mining NPs from genomic sequences are needed more than
ever to rejuvenate waning drug discovery pipelines, especially in light of the looming crisis
in antibiotic resistance207. The development of state-of-the-art high-throughput screening
platforms allow purified compounds or semi-pure extracts to be screened in hundreds of
assays with less material required than in previous decades208,209. The suite of NPs present
in nature is one of our most valuable natural resources, and we are now poised to more fully
explore the extent of its depth and diversity.
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Figure 1.
Natural product biosynthetic gene clusters. (a) Representation of the diversity of size and
complexity of NPs and their encoding gene clusters, including tyrvalin, a pyrazinone
virulence factor from skin-associated staphylococci210, platensimycin, a diterpenoid
antibiotic from soil-dwelling Streptomyces isolates211, pederin, a polyketide anticancer
agent produced by an uncultivated symbiont of the Paederus spp. beetles21, and bryostatin, a
macrocyclic lactone anticancer agent produced by a symbiont of a marine bryozoan212.
Approximate sizes of BGCs for select NPs (black), along with noteworthy examples of large
systems that have been built with synthetic DNA technology in wild type (red) or re-
designed (green) genetic architecture. (b) Widening gap of uncharacterized PKS enzymes
(grey) compared to biochemically characterized PKSs (black) since 2000 (data to 2010
reproduced from Wong and Khosla213; 2014 data point from Marnix Medema, personal
communication). Dashed line represents best fit to available data points. (c) Recent history
of DNA synthesis costs214 and the corresponding number of 50 kb gene clusters that could
be synthesized with $100k. Dotted lines project to the future along the same trajectory of the
past 15 years.Smanski et al. Page 22
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Figure 2.
Genetic refactoring. (a) Schematic outline of refactoring process, and (b) the streamlined
refactoring of homologous gene clusters by substituting coding sequences. New homologous
cluster and corresponding genetic parts are shown in green, and previously refactored cluster
and parts are shown in blue. Bold lines on chemical structures show conserved core scaffold
between two enediynes used as a hypothetical example. (c) Refactored epo gene cluster,
built into a two plasmid system. Extracted ion chromatogram shows production of
epothilones A and B from the refactored gene cluster introduced to M. xanthus (i), but not
from the wild-type host (ii)63.Smanski et al. Page 23
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Figure 3.
Genetic parts for controlling gene expression levels. (a) Characterization of genetic parts in
E. coli , including (from left to right), promoter variants30,31, ribosome insulators76,
bicistronic RBSs32, computationally designed RBSs34, and synthetic and natural
terminators33. (b) Genetic parts for engineering NP-producing organisms, including
promoter variants37–39, computationally designed RBSs36, and codon-optimized CDS
parts74.Smanski et al. Page 24
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Figure 4.
Exploiting refactored genetics for host transfer of multi-gene devices. (a) Schematic
representation of a DNA synthesis and assembly pipeline, wherein genetic parts are
constructed from synthetic oligonucleotides and then assembled into unique combinations.
(b) High-throughput library design of permuted gene clusters for antimalarial phosphonate
FR900098. Bar graph shows characterized titers from constructs selected from iterative
libraries, with successive libraries from left to right215. (c) Experimental design for
heterologous expression of ptn gene cluster and RT-PCR results for each operon in native
and wild-type hosts106. (d) The proposed platencin biosynthetic pathway, along with several
shunt metabolites isolated from a heterologous expression strain. Values show in red are
titers in heterologous host, while those shown in green are titers in the native producer. (e)
Illustration of behavior-matching via part replacement during host transfer. Graphs represent
empirical characterization of genetic parts in native host (green), and new host (red).
Landscape graphs show effect on gene clusters performance, as measured by titer of final
metabolite, in a multivariate system.Smanski et al. Page 25
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Figure 5.
Advanced regulation relevant to NP biosynthesis. Examples include (a) inducible promoters
for NP producing organisms216,217, (b) a mammalian genetic circuit responsive to a bacterial
metabolite218, (c) dynamic modeling results for a synthetic pathway for para-aminostyrene
production155, (d) a dynamic feedback/feedforward circuit for monitoring fatty acid ethyl
ester production in E. coli54, (e) a resource allocation system for controlling transcription of
a heterologous neurosporene operon in different hosts156, (f) a genetic reset timer for
controlled sedimentation in yeast160, and (g) multiplexed transcriptional control of the
violacein biosynthetic pathway using CRISPRi/CRISPRa172. For dynamic modeling
example (c), graphs show frequencies of expected yields for designs with static regulation
(top), dynamic regulation (middle), or for the particular pattern of dynamic regulation
pictured at left (bottom).Smanski et al. Page 26
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Figure 6.
Multiplexed genome editing with CRISPR/Cas9. (a) Minimal genome editing construct
design, including (i) sgRNA, (ii) S. pyogenes Cas9, and (iii) optional ‘repair fragment’.
Three routes to DNA repair are shown, including homologous recombination (HR, left),
alternative end-joining (AEJ, center), and non-homologous end-joining (NHEJ, center)195.
(b) Applications of CRISPR-mediated genome editing in Streptomyces . Graph at bottom
shows reported efficiencies for experiments grouped by application with background color
matching illustrations above. Protocol differences are labeled below graph, and data points
are colored according to published study (blue55, red57, green191). (c) Example of
multiplexed CRISPR editing for engineering mevalonate levels. Bar graph at bottom shows
editing efficiency (grey) and mevalonate levels (green), averaged across multiple different
combinations of gene deletions (number of combinations indicated in parentheses)188.Smanski et al. Page 27
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Figure 7.
Diverse applications of engineering NP biosynthesis. Structures of NPs are shown alongside
a representation of their BGCs with native producing organisms noted.Smanski et al. Page 28
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Atoms, Molecules and Photons | 57cd064ad1154708795c760532302a161a0d1c50 | page_0000 |
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Wolfgang Demt rö der
Atoms, Molecules
and Photons
An Introduction to Atomic-, Molecular-
and Quantum-Physics
W i t h6 7 7F i g u r e sa n d4 2T a b l e s
123 | 57cd064ad1154708795c760532302a161a0d1c50 | page_0002 |
Professor Dr. Wolfgang Demtröder
Universität KaiserslauternFB PhysikErwin-Schrödinger-Str. 4667663 [email protected]
ISSN 1868-4513 e-ISSN 1868-4521
ISBN 978-3-642-10297-4 e-ISBN 978-3-642-10298-1DOI 10.1007/978-3-642-10298-1Springer Heidelberg Dordrecht London New Y ork
Library of Congress Control Number: 2010935944
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Preface to the Second Edition
The first edition of this textbook had found a friendly acceptance. This second
edition does not change the concept of the representation which combines the ex-
perimental techniques for the investigation of atoms and molecules and their results
that have lead to the development of quantum physics. Some new developments
in laser physics and quantum optics have been inserted in Chapter 12 in order to
give the reader some ideas about the frontiers in these fields regarding experimental
techniques and physical insight.
This second edition represents a thoroughly revised version of the first edition,
which, unfortunately, contained a lot of errors and misprints. I am grateful to many
readers who have informed me about such errors and who offered corrections and
improvements of the representation for a better understanding. I am particularly in-
debted to Dr. Nico Dam, Raboud University Nijmegen, Netherland and Prof. Zamik
Rosenwaks, Ben Gurion University, Israel, who have sent me an extensive correction
list.
The author hopes, that this new edition will be well accepted and that critical
readers will send their comments or ideas about possible improvements.
I thank Dr. Th. Schneider, Springer Verlag for his continuous interest and
encouragement.
Kaiserslautern,
October 2010 Wolfgang Demtröder | 57cd064ad1154708795c760532302a161a0d1c50 | page_0004 |