Biology subjects

Chan, W.

Publications and source records attributed to Chan, W..

13 recordsLinked to original sources

bioRxiv2025

Cardiac Adaptations in the Cave Nectar Bat Eonycteris spelaea: Insights into Metabolic Resilience and Stress Response

AimsBats are unique mammals with remarkable adaptations, including powered flight, which demands significant energy expenditure. While previous studies have documented basic structural characteristics of bat hearts, a comprehensive understanding of their response to physiological stress remains unexplored. This study investigates the cardiac adaptations of the cave nectar bat Eonycteris spelaea to elucidate the mechanisms underlying their unique physiological capabilities. Methods and ResultsWe performed RNA sequencing to analyse the cardiac gene expression profile of E. spelaea and 6 other bat species in comparison to mouse and human hearts, revealing enriched transcriptomic signatures related to oxidative phosphorylation and fatty acid metabolism across multiple bat species. Metabolomic profiling compared acylcarnitine metabolites and tricarboxylic acid (TCA) cycle intermediates between bat and mouse hearts, indicating a distinct acylcarnitine profile and increased levels of TCA cycle intermediates in bats, suggesting enhanced metabolic capacity. Structural adaptations were assessed through anatomical and histological analyses on cardiac tissues, showing thicker left ventricular walls and increased vascular density in bats without pathological hypertrophy. Functional characteristics were evaluated using dobutamine stress echocardiography, demonstrating superior cardiac reserve in bats with significant increases in ejection fraction, stroke volume, and cardiac output under stress conditions. Additionally, isolated cardiomyocytes were treated with Angiotensin II (Ang II) to assess stress responses. Bat cardiomyocytes displayed resistance to Ang II-induced hypertrophy and mitochondrial dysfunction compared to mice, further highlighting their resilience to stress-induced damage. ConclusionThe unique adaptations observed in bat hearts, including enhanced metabolic pathways, structural remodelling, and cellular resilience contribute to their ability to meet the high energy demands of powered flight while maintain cardiac function under stress. These insights into bat cardiac physiology provide valuable information on cardioprotective mechanisms that could be applicable to other species. Translational PerspectiveBats exhibit remarkable cardiac adaptations that sustain the high energy demands of powered flight while resisting stress-induced damage. These insights into evolutionarily conserved cardioprotective mechanisms highlight potential therapeutic pathways for preventing heart failure, including resistance to hypertrophy and mitochondrial dysfunction under stress. Studying non-model organisms like bats may offer innovative approaches to enhance metabolic and stress resilience in human hearts, paving the way for translational research in cardiovascular disease management and treatment.

systems biology↗

bioRxiv2025

Notch-mediated regulation of β-Catenin-TCF activity instructs anteroposterior neuron positioning in C. elegans

Motor neuron positioning and organization along the neuroaxis is crucial for proper nervous system connectivity and function. In newly hatched C. elegans, the ventral nerve cord contains 22 motor neurons, divided into three classes (DD, DA, and DB), with their cell bodies showing a largely stereotypical positioning and sequential arrangement. However, the mechanisms controlling this precise positioning are not fully understood. Here, we uncover a left-right asymmetry in {beta}-catenin-TCF complex activity that controls motor neuron positioning. Loss of BAR-1/{beta}-catenin or POP-1/TCF causes a shift of motor neuron cell bodies toward the anterior, while loss of PRY-1/Axin shifts them toward the posterior. During embryonic ventral cord morphogenesis, BAR-1 expression is restricted to right-side motor neuron precursors through asymmetric Notch signaling, which promotes PRY-1 expression on the left to degrade BAR-1. Our findings highlight an atypical Notch-mediated regulation of Axin expression and reveal that left-right asymmetry during neuroaxis formation specifies anteroposterior motor neuron placement in the central nerve cord.

developmental biology↗

bioRxiv2024

Scalable cell-free production of active T7 RNA polymerase

The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a one-liter cell-free gene expression (CFE) reaction that achieves over 80% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags and agitation to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than 4 orders of magnitude reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.

synthetic biology↗

bioRxiv2024

Therapeutic modulation of ROCK overcomes metabolic adaptation of cancer cells to OXPHOS inhibition and drives synergistic anti-tumor activity

Genomic studies have identified frequent mutations in subunits of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A in non-small cell lung cancer. Previously, we and others have identified that SMARCA4-mutant lung cancers are highly dependent on oxidative phosphorylation (OXPHOS). Despite initial excitements, therapeutics targeting metabolic pathways such as OXPHOS have largely been disappointing due to rapid adaptation of cancer cells to inhibition of single metabolic enzymes or pathways, suggesting novel combination strategies to overcome adaptive responses are urgently needed. Here, we performed a functional genomics screen using CRISPR-Cas9 library targeting genes with available FDA approved therapeutics and identified ROCK1/2 as a top hit that sensitizes cancer cells to OXPHOS inhibition. We validate these results by orthogonal genetic and pharmacologic approaches by demonstrating that KD025 (Belumosudil), an FDA approved ROCK inhibitor, has highly synergistic anti-cancer activity in vitro and in vivo in combination with OXPHOS inhibition. Mechanistically, we showed that this combination induced a rapid, profound energetic stress and cell cycle arrest that was in part due to ROCK inhibition-mediated suppression of the adaptive increase in glycolysis normally seen by OXPHOS inhibition. Furthermore, we applied global phosphoproteomics and kinase-motif enrichment analysis to uncover a dynamic regulatory kinome upon combination of OXPHOS and ROCK inhibition. Importantly, we found converging phosphorylation-dependent regulatory cross-talk by AMPK and ROCK kinases on key RHO GTPase signaling/ROCK-dependent substrates such as PPP1R12A, NUMA1 and PKMYT1 that are known regulators of cell cycle progression. Taken together, our study identified ROCK kinases as critical mediators of metabolic adaptation of cancer cells to OXPHOS inhibition and provides a strong rationale for pursuing ROCK inhibitors as novel combination partners to OXPHOS inhibitors in cancer treatment.

cancer biology↗

bioRxiv2022

The Slingshot phosphatase 2 is required for acrosome biogenesis during spermatogenesis in mice

The acrosome is a membranous organelle positioned in the anterior portion of sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles towards nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knock-out (KO) mice and show that Slingshot phosphatase 2 (SSH2), a regulator of actin remodeling, is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with enhanced germ cell apoptosis and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.

developmental biology↗

bioRxiv2022

Viability Discrimination of Bacterial Microbiomes in Home Kitchen Dish Sponges via Propidium Monoazide Treatment

Dish sponges are known to support the survival and growth of human bacterial pathogens yet are commonly used by consumers to wash dishes and clean kitchen surfaces. Exposure to foodborne pathogens via sponge use may lead to foodborne illness, which is of particular concern among susceptible populations. Limitations associated with culture-independent or - dependent methods for bacterial community characterization have challenged the complete assessment of foodborne pathogen exposure risk presented by sponges. In this study, the bacterial microbiomes of five dish sponges were characterized to evaluate the presence of viable bacterial foodborne pathogens using propidium monoazide treatment, which is a novel approach in this medium. Total and viable sponge microbiomes were subsequently metataxonomically evaluated via targeted 16S rRNA sequencing. Select pathogen viability was confirmed using targeted selective enrichment. The comparison of total and viable sponge microbiome beta diversity indicated that sponge taxonomic abundance profiles did not vary significantly according to PMA treatment. The numbers of unique bacterial species (p-value = 0.0465) and unique bacterial foodborne pathogens (p-value = 0.0102) identified were significantly lower after PMA-treatment. A total of 20 unique bacterial foodborne pathogens were detected among sponge microbiomes regardless of PMA treatment. Three to six unique viable foodborne pathogens were identified in each sponge. Escherichia coli and Staphylococcus aureus were identified in all five viable sponge microbiomes evaluated. Viable E. coli were recovered from two of five sponges via targeted selective enrichment. These findings suggest that most sponge-associated bacterial communities may be viable and contain multiple viable bacterial foodborne pathogens. ImportanceBacterial pathogens may exist in the domestic kitchen environment, threatening both sanitation levels and the health of residents. Dish sponges are commonly used kitchen tools that can harbor foodborne pathogens as they present adequate conditions for the survival and growth of bacteria. Using a contaminated sponge may lead to foodborne illness through direct contact with pathogens or via cross-contamination with food or other surfaces. Although bacterial foodborne pathogens have been identified in sponges, previous limitations in methodology have prevented the complete understanding of sponge microbiomes. This study used a novel application of a chemical reagent coupled with targeted amplicon sequencing to identify sponge communities and differentiate between viable and non-viable bacteria. Insights into dish sponge microbiomes and potential risks of bacterial foodborne pathogen exposure can inform food safety education programs to aid in the prevention of home-acquired foodborne illness and cross-contamination events.

microbiology↗

bioRxiv2021

Gut microbiota and age shape susceptibility to clostridial enteritis in lorikeets under human care

BackgroundEnteritis is a common cause of morbidity and mortality in lorikeets that can be challenging to diagnose and treat. In this study, we examine gut microbiota in two lorikeet flocks with enteritis (Columbus Zoo and Aquarium - CZA; Denver Zoo - DZ). Since 2012, the CZA flock has experienced repeated outbreaks of enteritis despite extensive diet, husbandry, and clinical modifications. In 2018, both CZA and DZ observed a spike in enteritis. Recent research has revealed that the gut microbiota can influence susceptibility to enteropathogens. We hypothesized that a dysbiosis, or alteration in the gut microbial community, was making some lorikeets more susceptible to enteritis, and our goal was to characterize this dysbiosis and determine the features that predicted susceptibility. ResultsWe employed 16S rRNA sequencing to characterize the cloacal microbiota in lorikeets (CZA n = 67, DZ n = 24) over time. We compared the microbiota of healthy lorikeets, to lorikeets with enteritis, and lorikeets susceptible to enteritis, with "susceptible" being defined as healthy birds that subsequently developed enteritis. Based on sequencing data, culture, and toxin gene detection in intestinal contents, we identified Clostridium perfringens type A (CZA and DZ) and C. colinum (CZA only) at increased relative abundances in birds with enteritis. Histopathology and immunohistochemistry further identified the presence of gram-positive bacilli and C. perfringens, respectively, in the necrotizing intestinal lesions. Finally, using Random Forests and LASSO models, we identified several features (young age and the presence of Rhodococcus fascians and Pseudomonas umsongensis) associated with susceptibility to clostridial enteritis. ConclusionsWe identified C. perfringens type A and C. colinum associated with lorikeet necrohemorrhagic enteritis at CZA and DZ. Susceptibility testing of isolates lead to an updated clinical treatment plan which ultimately resolved the outbreaks at both institutions. This work provides a foundation for understanding gut microbiota features that are permissive to clostridial colonization and host factors (e.g. age, prior infection) that shape responses to infection.

microbiology↗

bioRxiv2021

Sex- and context-dependent effects of acute isolation on vocal and non-vocal social behaviors in mice

Humans are extraordinarily social, and social isolation has profound effects on our behavior, ranging from increased social motivation following short periods of social isolation to increased anti-social behaviors following long-term social isolation. Mice are frequently used as a model to understand how social isolation impacts the brain and behavior. While the effects of chronic social isolation on mouse social behavior have been well studied, much less is known about how acute isolation impacts mouse social behavior and whether these effects vary according to the sex of the mouse and the behavioral context of the social encounter. To address these questions, we characterized the effects of acute (3-day) social isolation on the vocal and non-vocal social behaviors of male and female mice during same-sex and opposite-sex social interactions. Our experiments uncovered pronounced effects of acute isolation on social interactions between female mice, while revealing more subtle effects on the social behaviors of male mice during same-sex and opposite-sex interactions. Our findings advance the study of same-sex interactions between female mice as an attractive paradigm to investigate neural mechanisms through which acute isolation enhances social motivation and promotes social behavior.

animal behavior and cognition↗

bioRxiv2021

DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation

T cell receptor clustering plays a key role in triggering cell activation, but the relationship between the spatial configuration of clusters and elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami-based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by non-stimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands and translates that information into distinct signaling dynamics, and provide insights into engineering new immunotherapies.

cell biology↗

bioRxiv2020

Tmed2 regulates Smoothened trafficking and Hedgehog signalling

Hedgehog (HH) signalling plays a key role in embryonic pattering and stem cell differentiation. Compounds that selectively bind Smoothened (SMO) can induce cell death in mouse embryonic stem cells (ESCs). Here we perform a genetic screen in haploid ESCs and discover that SMO inhibits a cell death pathway that resembles dissociation induced death of human ESCs and Anoikis. In mouse ESCs, SMO acts through a G-protein coupled mechanism that is independent of GLI activation. Our screen also identifies the Golgi proteins Tmed2 and Tmed10. We show that TMED2 binds SMO and controls its abundance at the plasma membrane. In neural differentiation and neural tube pattering Tmed2 acts as a repressor of HH signalling strength. We demonstrate that the interaction between SMO and TMED2 is regulated by HH signalling suggesting SMO release form the ER-Golgi is critical for controlling G-protein and GLI mediated functions of mammalian HH signalling.

cell biology↗

bioRxiv2020

Efficient immune cell genome engineering with improved CRISPR editing tools

CRISPR (clustered regularly interspaced short palindromic repeats)-based methods have revolutionized genome engineering and the study of gene-phenotype relationships. However, modifying cells of the innate immune system, especially macrophages, has been challenging because of cell pathology and low targeting efficiency resulting from nucleic acid activation of sensitive intracellular sensors. Likewise, lymphocytes of the adaptive immune system are largely refractory to CRISPR-enhanced homology-directed repair (HDR) due to inefficient or toxic delivery of donor templates via transient transfection methods. To overcome these challenges and limitations, we developed three improved methods for CRISPR-based genome editing using a hit-and-run transient expression strategy to minimize off-target effects and generate more precise genome editing. Overall, our enhanced CRISPR tools and strategies designed to tackle both murine and human immune cell genome engineering are expected to be widely applicable not only in hematopoietic cells but also other mammalian cell types of interest. All animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional Animal Care and Use Committee.

genomics↗

bioRxiv2018

Transcriptomic responses of the marine cyanobacterium Prochlorococcus to viral lysis products

Marine phytoplankton contributes to about one half of global primary production, and a significant proportion of their photosynthetically fixed organic carbon is released after viral infection as dissolved organic matter (DOM). This DOM pool is known to be consumed by heterotrophic microorganisms; however, its impact on the uninfected co-occurring phytoplankton remains largely unknown. Here, we conducted transcriptomic analyses to study the effects of viral lysis products on the unicellular cyanobacterium Prochlorococcus, which is the most abundant photosynthetic organism on Earth. While Prochlorococcus growth was not affected by viral lysis products, many tRNAs increased in abundance, which was also seen after amino acid addition, suggesting that amino acids are one of the compounds in viral lysis products that affected the expression of tRNA genes. The decreased transcript abundances of N metabolism genes also suggested that Prochlorococcus responded to organic N compounds, consistent with abundant amino acids in viral lysis products. The addition of viral lysis products to Prochlorococcus reduced the maximum photochemical efficiency of photosystem II and CO2 fixation while increased its respiration rate, consistent with differentially expressed genes related to photosynthesis and respiration. One of the highest positive fold-changes was observed for the 6S RNA, a non-coding RNA functioning as a global transcriptional regulator in bacteria. The high level of 6S RNA might be responsible for some of the observed transcriptional responses. Taken together, our results revealed the transcriptional regulation of Prochlorococcus in response to viral lysis products and suggested its metabolic potential to utilize organic N compounds.\n\nImportancePhotosynthetic microorganisms called phytoplankton are abundant in the oceans and contribute to about one half of global CO2 fixation. Phytoplankton are frequently infected by viruses and after infection their organic carbon is released into the ocean as dissolved organic matter (DOM). Marine DOM is important for the marine food web because it supports the growth of heterotrophic microorganisms. However, the impact of viral DOM on the uninfected phytoplankton is largely unknown. In this study, we conducted transcriptomic analyses and identified many differentially expressed genes when viral DOM was added to the marine cyanobacterium Prochlorococcus. One effect of viral DOM is that the carbon fixation of Prochlorococcus was reduced by ~16%, which might affect carbon cycling in the worlds oceans since Prochlorococcus is the most abundant photosynthetic organism on Earth.

microbiology↗

bioRxiv2018

An integrative systems biology and experimental approach identifies convergence of epithelial plasticity, metabolism, and autophagy to promote chemoresistance

The evolution of therapeutic resistance is a major cause of death for patients with solid tumors. The development of therapy resistance is shaped by the ecological dynamics within the tumor microenvironment and the selective pressure induced by the host immune system. These ecological and selective forces often lead to evolutionary convergence on one or more pathways or hallmarks that drive progression. These hallmarks are, in turn, intimately linked to each other through gene expression networks. Thus, a deeper understanding of the evolutionary convergences that occur at the gene expression level could reveal vulnerabilities that could be targeted to treat therapy-resistant cancer. To this end, we used a combination of phylogenetic clustering, systems biology analyses, and wet-bench molecular experimentation to identify convergences in gene expression data onto common signaling pathways. We applied these methods to derive new insights about the networks at play during TGF-{beta}-mediated epithelial-mesenchymal transition in a lung cancer model system. Phylogenetics analyses of gene expression data from TGF-{beta} treated cells revealed evolutionary convergence of cells toward amine-metabolic pathways and autophagy during TGF-{beta} treatment. Using high-throughput drug screens, we found that knockdown of the autophagy regulatory, ATG16L1, re-sensitized lung cancer cells to cancer therapies following TGF-{beta}-induced resistance, implicating autophagy as a TGF-{beta}-mediated chemoresistance mechanism. Analysis of publicly-available clinical data sets validated the adverse prognostic importance of ATG16L expression in multiple cancer types including kidney, lung, and colon cancer patients. These analyses reveal the usefulness of combining evolutionary and systems biology methods with experimental validation to illuminate new therapeutic vulnerabilities.

cancer biology↗