Laterally Orienting C. elegans Using Geometry at Microscale for High-Throughput Visual Screens in Neurodegeneration and Neuronal Development Studies

C. elegans is an excellent model system for studying neuroscience using genetics because of its relatively simple nervous system, sequenced genome, and the availability of a large number of transgenic and mutant strains. Recently, microfluidic devices have been used for high-throughput genetic screens, replacing traditional methods of manually handling C. elegans. However, the orientation of nematodes within microfluidic devices is random and often not conducive to inspection, hindering visual analysis and overall throughput. In addition, while previous studies have utilized methods to bias head and tail orientation, none of the existing techniques allow for orientation along the dorso-ventral body axis. Here, we present the design of a simple and robust method for passively orienting worms into lateral body positions in microfluidic devices to facilitate inspection of morphological features with specific dorso-ventral alignments. Using this technique, we can position animals into lateral orientations with up to 84% efficiency, compared to 21% using existing methods. We isolated six mutants with neuronal development or neurodegenerative defects, showing that our technology can be used for on-chip analysis and high-throughput visual screens

Association between pre-biologic T2-biomaker combinations and response to biologics in patients with severe asthma

Funding This study was conducted by the Observational and Pragmatic Research Institute (OPRI) Pte Ltd and was partially funded by Optimum Patient Care Global (OPCG) and AstraZeneca Ltd. No funding was received by the OPRI for its contribution. The International Severe Asthma Registry (ISAR) is operated by OPCG and co-funded by OPCG and AstraZenecaPeer reviewe

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

On-chip phenotypic screening and characterization of C. elegans enabled by microfluidics and image analysis methods

Since its introduction in 1960's, the model organism Caenorhabditis elegans has played a crucial role towards scientific discoveries because of its relatively simple anatomy, conserved biological mechanisms, and mapped genome. The organism also has a rapid generation time and produces a large number of isogenic progeny, making C. elegans an excellent system for conducting forward genetic screens. Conventional screening methods, however, are labor intensive and introduce potential experimental bias; typically, large-scale screens can take months to years. Thus, automated screening and characterization platforms can provide an opportunity to overcome this bottleneck. The objective of this thesis is to develop tools to perform rapid phenotypical characterization of C. elegans to enable automated genetic screening systems for neural development. To achieve this goal, I developed methods to increase throughput of worm handling using microfluidic devices and demonstrate software modules to phenotype unknown mutants using quantitative and morphological image analysis methods. Microfluidic devices are constructed from PDMS using established soft-lithography techniques. The emphasis on the simplification of existing designs greatly facilitates the adoption of our developed systems by other scientists. This thesis also includes image processing modules using various techniques to determine animal phenotypes. For example, we adapted standard thresholding methods to detect animal motor neurons, developed a modified granulometry algorithm to rapidly characterize large numbers of lipid droplets in 3D, and developed a probability model to determine neuronal process morphology. This work is significant because it increases current capabilities of screening small animals with morphological phenotypes by enhancing throughput and reducing human bias. Genes or gene functions that can be discovered using these methods can further elucidate mechanisms relevant to neural development, degeneration, maintenance, and function; these discoveries in turn can facilitate discoveries of potential therapeutic strategies for human neurological diseases.Ph.D

L1 phenotype characterization.

<p>(A–G) Proportion of animals in a population with at least one incidence of each of the independent defects: guidance, handedness, and missing defects seen for wild-type (<i>juIs76</i>) and newly isolated mutants. Asterisks indicate statistical significance of phenotype compared to wild-type. Single, double, and triple asterisks indicate <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001 respectively. Statistical significance determined using chi-squared test. Number of animals examined for each allele labeled in graph. Error bars represent standard error of proportion.</p

Microfluidic device to passively orient <i>C. elegans</i> for visual screening.

<p>(A) Device used for orienting, imaging, and sorting animals. Flow layer is shown in green with black text, valve control layer in red with white text. LD is loading valve, MT is channel for mutant output, and WT is channel (black) and valve (white) for wild-type output. IMG is imaging valve. Not labeled are the mutant valve (right of mutant channel) and flush channel (green area above red box). Input and flush channel fluid flow are controlled off-chip. Imaging area is indicated by red dashed box. Scale bar is 800 µm. (B) Device schematic of valve state during worm loading. Worm is driven into imaging area using positive input pressure to induce fluid flow. Loading valve is not actuated, allowing fluid flow. Imaging valve is actuated to prevent worm from exiting imaging area. Wild-type valve is not actuated to assist in worm loading and to provide an exit should an animal slip past the imaging valve. Mutant valve is actuated to prevent animals from entering mutant output. (C) Device schematic of valve state during analysis. All valves are actuated and input pressure is cut off halting fluid flow. (D) Device schematic of valve state during worm sorting. Example for wild-type sorting is shown. Worm is driven out of the imaging area by positive pressure from the flush channel. Loading valve is actuated to prevent any other animals from entering the imaging area. Imaging valve is not actuated to allow worm exit. Wild-type valve is not actuated to allow worm exit into wild-type output. Mutant valve is actuated to prevent animals from entering mutant output. Mutant and wild-type valve state is reversed when mutant sorting is performed. Fluid flow direction indicated by white arrows and is not proportional to size of arrow. Non-filled and filled red boxes indicate non-actuated and actuated valves respectively.</p

Morphology of wild-type and defective D-type motor neuron commissures.

<p>(A) Full body image of adult worm. Scale bar is 25 µm. (B) Expanded view of area within dashed box in panel A. Ventral nerve cord (VNC) is distinguished by presence of neuronal cell bodies. Dorsal nerve cord labeled DNC. Scale bar is 25 µm. (C) Representative image of panel B. Black arrows label neuronal commissures while black arrowheads identify neuronal cell bodies. Respective phenotypes characterized by: (D) commissure never reaching dorsal nerve cord; (E) commissure running along the opposite side of the animal's body (left, colored yellow); (F) absent commissure; (G) additional commissure present; (H) two commissures crossing over each other; (I) two commissures entering the dorsal nerve cord or leaving the ventral nerve cord together, they may also partially fasciculate; (J) bifurcating commissure; (K) neighboring commissures joined by a process; (L) neurite with length less than half nematode width; (M) an absence of GFP expression along either dorsal or ventral nerve cords; (N) break in GFP expression in a commissure. Scale bar is 25 µm. Representative image of phenotype shown beneath each photo accompanied with illustrative phenotype symbol. (O) Proportion of animals in a population with at least one incidence of each independent defect. Error bars represent standard error of proportion.</p

Adult phenotype characterization.

<p>(A–G) Proportion of animals in a population with at least one incidence of each of the independent defects: guidance, handedness, missing, and gap for wild-type (<i>juIs76</i>) and newly isolated mutants. Asterisks indicate statistical significance of phenotype compared to wild-type. Single, double, and triple asterisks indicate <i>p</i><0.05, <i>p</i><0.01, and <i>p</i><0.001, respectively. Pound signs indicate statistical significance of phenotype compared between <i>a073</i> and <i>a074</i>. Double and triple pound signs indicate <i>p</i><0.01 and <i>p</i><0.001, respectively. Statistical significance determined using chi-squared test. Number of animals examined for each allele labeled in graph. Error bars represent standard error of proportion.</p

Effect of curved channel on animal orientation.

<p>(A) Zoomed in view of imaging area shown by dashed red box from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035037#pone-0035037-g001" target="_blank">Figure 1A</a>. White arrow indicates device radius of curvature (RoC) from arc center to outer edge. Dashed line indicates length (L) between loading and imaging valves. Scale bar is 200 µm. (B) Frequency of lateral nematode orientation for various channel geometries with standard error of proportion. Triple asterisk indicates statistical significance compared to straight channel designs (<i>p</i><0.001 determined using chi-squared test). S represents straight channel, while remaining labels indicate the 105 µm, 125 µm, and 145 µm RoC devices respectively. L for all devices is 700 µm. (C) Nematode oriented laterally in curved channel device (both nerve cords visible). Commissures present on different focal plane are obscured. (D) 3-D model of animal body section and microscope objective (viewpoint reference) showing nerve cord placement for a laterally oriented animal. (E) Nematode in a non-lateral body orientation as observed when loading animals into straight channel devices; DNC not visible due to animal orientation. Animal is within the same field of view as seen in panel C. Arrowheads for images (C) and (E) indicate ventral nerve cord (VNC) determined by placement of VD and DD motor neuron cell bodies. Arrow indicates dorsal nerve cord (DNC). Scale bars are 100 µm. Transgene marker for all fluorescent images is <i>juIs76(Punc-25::GFP</i>). (F) 3-D model of animal body section and microscope objective for non-lateral oriented animal. Model diagrams (D) and (F) not drawn to scale. Red lines illustrate dorso-ventral axis. Green spheres represent DD and VD neuron cell bodies.</p