Biology subjects

Tam, C.

Publications and source records attributed to Tam, C..

12 recordsLinked to original sources

bioRxiv2025

Dual schema of allergens reveals molecular and evolutionary signatures

Allergies affect billions of people worldwide, posing a substantial challenge to global health because of its elusive molecular determinants. Leveraging high-quality protein structures, we revisit why allergenic proteins trigger allergies, whereas some of their structural analogues do not. We categorize allergenic protein folds into similar (SAP) and dissimilar (DAP) folds on the basis of their similarity to human proteins, establishing a dual schema that reveals distinct molecular and evolutionary patterns. Here, we show that compared with structurally similar, nonallergenic protein (SNAP) folds, SAP folds are ubiquitous across kingdoms and rich in B- and T-cell epitopes. DAP folds map to a unique sequence-structure-function space and are evolutionarily underrepresented and taxonomically restricted. The persistent divergence of DAP folds from animal proteins suggests their intermittent immune encounters. In contrast, ubiquitous SAP folds require continuous immune sorting from their SNAP analogues. This dichotomy provides a new lens for studying the evolution of host immune responses in allergic reactions. Our classifier, AllerX, is trained on these patterns and predicts unseen APs with state-of-the-art accuracy. This work, which links the structural, immunological, and evolutionary aspects of allergenic proteins, establishes a novel framework for enhancing diagnostics and food safety. SignificanceAllergies, a global health challenge, cause life-threatening immune reactions. Our theory links protein foreignness to immunogenicity, but its structural definition based on similarity to human proteins remains undefined. Utilizing massive protein structure resources, we grouped allergenic protein folds into similar (SAP) or dissimilar (DAP) to human proteins. We reveal strong contrasts in molecular and evolutionary signatures: DAP folds are taxonomically restricted but have biased, source-specific distribution among kingdoms. SAP folds show prominent, orientation-specific B-cell epitopes compared to their non-allergenic structural analogs. We also show fold-independent T-cell epitope linkage between SAP and DAP folds as a converged allergenicity signal. This dichotomy enhances cross-reactivity tracing in food safety incidents and establishes a foundation for studying host-factor evolution of protein allergy.

bioinformatics↗

bioRxiv2025

Topical Formulation of Repurposed FDA-Approved Compounds Inhibits Pseudomonas aeruginosa ExoU and Improves Corneal Infection Outcomes

Microbial keratitis, infection of the cornea, cause by ExotoxinU (ExoU) strains of Pseudomonas aeruginosa have a poor clinical outcome and response to antimicrobials. ExoU, a phospholipase, is secreted directly into host cells, causing their lysis. A screen of 3,034 FDA-approved compounds identified zinc pyrithione (Zp), bismuth subcitrate (Bis), and polymyxin B (Pol) as lead inhibitors of ExoU selectively inhibiting it without affecting human PLA2s or bacterial viability. The compounds have distinct inhibitory mechanisms: Zp disrupted ExoU oligomerization and protein stability; Bis impaired phosphatidylinositol 4,5-bisphosphate-dependent membrane association; Pol directly inhibited catalysis via its lipid-peptide architecture. Compound combinations enhanced ExoU inhibition in vitro to nanomolar concentrations. In mammalian cells, Bis and Pol promoted lysosomal trafficking and degradation of ExoU. Using a high-throughput microscopy platform, we screened 53 P. aeruginosa keratitis isolates, confirming broad efficacy of these inhibitors in exoU strains. Therapeutic efficacy was evaluated in ex vivo porcine corneas, Galleria mellonella, and in vivo mouse keratitis models. Topical delivery of ExoU inhibitors, particularly in combination, significantly reduced corneal opacity, ulceration, and stromal damage in porcine corneas without affecting bacterial load. In Galleria, compound combinations significantly maintained larval health, improved larval survival and delayed mortality. In a mouse eye infection model, combinatorial treatment reduced disease severity and preserved tissue viability without altering bacterial burden. These findings validate ExoU as a druggable virulence factor and support the repurposing of these compounds as an anti-virulence strategy for the treatment of P. aeruginosa infections in humans and veterinary medicine.

microbiology↗

bioRxiv2025

HuBIE: The Human Blood Immunome Encyclopedia Of TCRs and BCRs in Bloodstream Infections and Cancer

T and B cells are central to adaptive immunity, where they identify and neutralize foreign antigens and cancer neo-antigens. Large-scale elucidation of T- and B-cell receptors (TCRs and BCRs) through immune-repertoire sequencing promises novel diagnostics, prognostic markers, and therapeutic strategies. However, progress is hampered by small cohort sizes, a lack of real-world patient diversity, and heterogeneous sample processing, impeding cross-study comparability. To overcome these limitations, here we introduce the Human Blood Immunome Encyclopedia (HuBIE), comprising immune-repertoire data from 2,614 samples collected from 1,941 participants. The cohort includes a range of bloodstream infections, several cancer types, and control participants, with many individuals providing longitudinal samples. We employed Roches immune receptor Primer Extension Target Enrichment (immunoPETE) platform to perform simultaneous targeted sequencing of T-cell receptor {beta} chains (TRB), T-cell receptor {delta} chains (TRD), and immunoglobulin heavy chains (IGH), thereby profiling TCRs and BCRs in all participants. We provide a comprehensive description of immune-repertoire diversity in cancer and bloodstream infections and examine variations across demographic variables such as age and race. We find significant differences in TRB and IGH composition across ethnic groups, and show that the fall in repertoire diversity with age follows distinct patterns for TRB, TRD, and IGH and is accompanied by age-related differences in VJ gene usage. Finally we demonstrate that greater immunological diversity is associated with improved survival but only for elderly participants. HuBIE thus constitutes a valuable resource for the immune-repertoire community, enabling large-scale mapping of the human immunome to accelerate development of diagnostics, prognostic biomarkers, and innovative therapeutic strategies.

immunology↗

bioRxiv2025

Uncovering functional insights into human pathogenic variants in CDK19 using Drosophila models

Heterozygous missense variants in CDK19 have been found in patients diagnosed with Developmental and epileptic encephalopathy-87 (DEE87) who present with global developmental delay, intellectual disability and other muscular and neurological deficiencies. Two missense variants in CDK19, Y32H and T196A, were first proposed to be dominant negative in nature based on experiments in a Drosophila model. Subsequently, another group proposed that Y32H is a gain of function based on elevated kinase activity. We present a detailed evaluation of the activity and functionality of these dominant variants in several contexts in fly models of DEE-87 in which endogenous cdk8, the fly ortholog of human CDK8 and CDK19, is knocked down while we overexpressed the human genes. Depletion of Drosophila cdk8 causes thicker muscle myofibrils, fused mitochondria, and climbing defects. The expression of human CDK19WT in a fly cdk8-RNAi background rescues these defects, highlighting functional conservation. To investigate functional differences between the variants, we compared effects of variants to expression of wildtype CDK19. Ubiquitous expression of Y32H can rescue the cdk8 knockdown phenotype through a gain of function compensatory effect, while T196A is unable to do so through a possible reduction in kinase activity. We found that supplementation of the antioxidant drug, N-acetylcysteine amide (NACA), rescues phenotypes of only the T196A adult flies, illustrating a divergence in variant functionality. Our studies in flies allowed us to assay these variants in numerous contexts to gain further insight into their mechanism and obtain translational knowledge to apply back to human health.

genetics↗

bioRxiv2025

miR-190 is a Key Regulator in Establishing Cell Polarity and Specification in the Drosophila Nervous System

Asymmetric cell division generates cellular diversity in developing tissues, particularly in the CNS. In Drosophila neuroblasts, this process relies on polarity complexes and fate determinants, yet its molecular regulation remains unclear. Here, we identify miRNA-190 as a key regulator of neuroblast polarity and differentiation. Single-cell RNA sequencing and transcriptome analysis reveal that miR-190 deficiency disrupts CNS cell populations, reducing neurons while increasing neural progenitors and glia. Mechanistically, miR-190 is required for proper localization of the Par complex and basal determinants during mitosis. In miR-190 mutants, these factors mislocalize, leading to defective polarity and fate specification in embryonic neuroblast. qPCR analysis shows that miR-190 targets RhoGAP, which modulates Cdc42 activation and Par-6, crucial factors in neuroblast polarity. We propose a model in which miR-190 ensures proper Cdc42 activation and polarity establishment by targeting transcripts for degradation. miR-190 has been implicated in various cancers, and our findings provide a mechanistic framework for understanding miR-190s roles in tumorigenesis and its broader involvement in metabolic diseases.

cell biology↗

bioRxiv2024

eDNA sampling systems for salmon ecosystem monitoring

Environmental DNA (eDNA) is transforming the way aquatic ecosystems are monitored and managed by scientists, resource managers, ENGOs, First Nations communities, and citizen scientists alike. However, the lack of sampling systems enabling high filtration volumes and rapid sample collection in the field have thus far hindered broad scale eDNA studies in the ocean specifically for small and medium scale organizations. To overcome these challenges, several modular water sampling systems that utilize hollow-membrane filtration cartridges were developed by RKS laboratories and tested by the Fisheries and Oceans, Canada, Molecular Genetics Laboratory. Compared to Sterivex filters, an industry standard for eDNA filtration, the hollow-membrane filtration cartridges allowed for a six-fold increase in filtration volume and threefold increase in filtration speed. The field sampling systems, which combine pumps, a programmable controller, an air pump, an ozone generator, and up to eight filters at once, enabled efficient direct eDNA filtration from diverse aquatic environments, from creeks to the open ocean. To evaluate ease of deployment, we present the results of a three day workshop where technical staff of an Indigenous resource management organization, without any prior knowledge in eDNA sampling, were trained and performed independent eDNA sample collection. The samples were analyzed by metabarcoding and qPCR to reveal the distributions of salmon and other species co-occurring in salmon ecosystems, from large ephemeral predators, to the planktonic prey of salmon, even including their pathogens. In this example study, we further observed a substantial shift in community composition in the vicinity of aquaculture facilities where marine species associated with aquaculture feed were detected in freshwater at high relative abundance. This study demonstrates how these sampling systems provide an efficient entry point for small and medium scale organizations to utilize eDNA to fulfill their research and monitoring objectives.

genomics↗

bioRxiv2024

Single-cell analysis of an engineered organoid-based model of pancreatic cancer identifies hypoxia as a contributing factor in the determination of transcriptional subtypes

Pancreatic ductal adenocarcinoma (PDAC) is a high-mortality cancer characterized by its aggressive, treatment-resistant phenotype and a complex tumour microenvironment (TME) featuring significant hypoxia. Bulk transcriptomic analysis has identified the "classical" and "basal-like" transcriptional subtypes which have prognostic value in PDAC; however, it remains unclear how microenvironmental heterogeneity contributes to the expression of these transcriptional signatures. Here, we used single cell transcriptome analysis of the organoid TRACER platform to explore the effect of oxygen and other microenvironmental gradients on PDAC organoid cells. We found that the microenvironmental gradients present in TRACER significantly impact the distribution of organoid transcriptional phenotypes and the enrichment of gene sets linked to cancer progression and treatment resistance. More significantly, we found that microenvironmental gradients drive changes in the expression of the classical and basal-like transcriptional subtype gene signatures. This effect is likely dominated by the oxygen gradients in TRACER, as hypoxia alone induced decreases in the expression of classical marker GATA6 at both the gene and protein level in PDAC cells. This work suggests that hypoxia contributes to determining transcriptional subtypes in PDAC and broadly underscores the importance of considering microenvironmental gradients in organoid-based transcriptomic studies of PDAC.

cancer biology↗

bioRxiv2024

Epithelial cytokeratin 6a restricts secretory autophagy of proinflammatory cytokines by interacting with Sec16A

Epithelial cells form a crucial barrier against harmful microbes and inflammatory stimuli. Restraining inflammatory responses at the corneal barrier is necessary for avoiding sight-threatening tissue damage. Yet, epithelial cell-intrinsic mechanisms that dampen inflammation are largely unexplored. Keratin 6a (K6a) is a common type II cytokeratin highly expressed in corneal and other stratified epithelial cells. In a mouse model of sterile corneal inflammation, K6a knockout mice exhibit disease exacerbation. Here, we investigated cell-intrinsic mechanisms by which cytoplasmic K6a curbs corneal inflammation. We stimulated wild-type (WT) and K6a siRNA-knockdown (K6a-KD) human corneal epithelial (hTCEpi) cells with inflammatory P. aeruginosa culture supernatant. Our results showed that, under both basal and inflammatory conditions, K6a-KD cells secreted higher levels of cytokines and chemokines (IL-1, IL-6, IL-8, CXCL1, CCL20) as compared to WT cells. K6a-KD cells also had increased level of LC3-II, a marker for autophagosomes, while autophagic degradation of SQSTM1/p62 remained unchanged. In K6a-KD cells, the majority of LC3-II puncta were associated with non-acidified autophagosomes rather than acidified autolysosomes. Upon stimulation, IL-8 was found to co-localize with LC3-II by confocal microscopy. Mechanistically, mass spectrometric analysis of K6a immunoprecipitates identified Sec16A, a protein involved in secretory autophagy, as an interacting partner of K6a. Further experiments showed that knocking down key proteins involved in autophagosome formation (ATG5) and the secretory autophagy process (Sec16A, GRASP55, Rab8) abolished the augmentative effect of K6a-KD on cytokine and chemokine secretion. These findings reveal a novel repressive role of K6a in secretory autophagy-mediated proinflammatory cytokine secretion and provide new insights into cell-intrinsic mechanisms of inflammation control at epithelial barriers.

cell biology↗

bioRxiv2023

Keratin 6a attenuates Toll-like receptor-triggered proinflammatory response in corneal epithelial cells by suppressing ELKS/IKKϵ-dependent activation of NF-κB

The corneal epithelium at the ocular surface is constantly exposed to the environment and represents the first line of defense against infection, mechanical injury or chemical irritation. Through TLR-mediated recognition of pathogen- and damage-associated molecular patterns, it engages in direct antimicrobial responses and alerts the immune system on intruder and tissue damage by secreting pro-inflammatory and chemotactic cytokines that promote immune cell infiltration. How the corneal epithelium downregulates TLR signaling is unclear, yet it highly expresses keratin 6a (K6a), a cytoskeletal protein that has emerged to play essential regulatory roles in corneal innate immune response. Here we report that mice harboring genetic deletion of K6a are more susceptible to developing bacterial keratitis with unresolved corneal opacification and higher bacterial load. Such disease phenotype is caused by the increased pro-inflammatory cytokine and chemokine secretions from the K6a-null corneal epithelium, which further promotes the infiltration of immune cells and their associated pro-inflammatory response. Using human corneal epithelial cells immortalized by telomerase reverse transcriptase (hTCEpi cells), we demonstrated that knocking down K6a enhances NF-{kappa}B/ RelA-dependent cytokine and chemokine expression. Moreover, proteomic screen reveals that K6a interacts with ELKS, a critical NEMO-binding scaffold that links between canonical IKK/{beta} and the principal cytoplasmic inhibitor of RelA, i.e. I{kappa}B., to promote its phosphorylation and degradation. Surprisingly, K6a does not antagonize any of these canonical NF-{kappa}B signaling events. Instead, we found that ELKS in addition to canonical IKKs interacts with the atypical IKK member IKK{varepsilon}. Furthermore, knockdown of K6a in hTCEpi cells promotes ELKS-dependent phosphoactivation of IKK{varepsilon}, which in turn phosphorylates and activates RelA. Our study thus demonstrated an unexpected role of cytosolic K6a as a novel negative regulator of TLR/NF-{kappa}B signaling in preventing excess proinflammatory cytokine and chemokine expressions. It further highlighted the functional importance of ELKS as a common signaling scaffold for both canonical and atypical IKK-dependent activation of NF-{kappa}B in corneal epithelial cells. Using both IKK classes other than only canonical IKKs for TLR/NF-{kappa}B induction as in other cell types including myeloid immune cells suggest that the cornea epithelium is more flexible in modulating its inflammatory response, which could greatly minimize corneal damage while preserving its essential functions for barrier protection and light refraction.

immunology↗

bioRxiv2022

A deficiency screen of the 3rd chromosome for dominant modifiers of the Drosophila ER integral membrane protein, Jagunal

The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the Endoplasmic Reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, we performed a dominant modifier screen of the 3rd chromosome for enhancers and suppressors of this Jagn RNAi-induced rough eye phenotype. We screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn RNAi phenotype. Based on the functions of the genes covered by the deficiencies, we identified genes that displayed a suppression or enhancement of the Jagn RNAi phenotype. These include Division Abnormally Delayed (Dally), an heparan sulfate proteoglycan, the {gamma}-secretase subunit Presenilin, and the ER resident protein Sec63. Based on our understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis.

genetics↗

bioRxiv2021

Simultaneous Control of Infection and Inflammation by Keratin-Derived Antimicrobial Peptides (KAMPs) Targeting TLRs and Co-receptors

The use and the timing of initiation of steroids for controlling unwanted infectious inflammation are major clinical dilemmas due to their possible adverse effects including delayed microbial clearance and wound healing. Compounding this difficulty is the continued emergence of drug-resistant bacteria; yet anti-infective strategies aiming at augmenting inflammatory responses to facilitate bacterial killing cannot be used to treat infections involving vulnerable tissues. As is the case with bacterial keratitis, excessive inflammation jeopardizes corneal transparency leading to devastating vision loss. Hence, a two-pronged remedy possessing both anti-infective and anti-inflammatory properties would be helpful for tackling antibiotic resistance and enabling prompt inflammation control at once. Using murine primary neutrophils, macrophages and sterile corneal inflammation models, we found that non-toxic and pro-healing human keratin 6a-derived antimicrobial peptides (KAMPs) with a native 10-or 18-amino-acid sequence suppress LTA- and LPS-induced NF-[kcy]B and IRF3 activation, proinflammatory cytokine production, as well as phagocyte recruitment, independently of their bactericidal function. Mechanistically, direct binding of KAMPs to cell surface TLR2 and TLR co-receptors CD14 and MD-2 not only blocks their bacterial ligand docking sites, but also reduces cell surface availability of TLR2 and TLR4 through promotion of receptor endocytosis. Benefitting from the dual functions of topical KAMPs, experimental bacterial keratitis caused was effectively prevented or controlled, as evidenced by significant reductions of corneal opacification and inflammatory cell infiltration in addition to enhanced bacterial clearance. These findings reveal multiple TLR-targeting activities of KAMPs and demonstrate their therapeutic potential as a multifunctional drug for managing sterile and infectious inflammatory diseases. One Sentence SummaryBifunctional native keratin peptides allow concurrent alleviation of inflammation and infection to avoid functional damages in vulnerable tissues.

immunology↗

bioRxiv2019

Identification of type 2 diabetes loci in 433,540 East Asian individuals

Meta-analyses of genome-wide association studies (GWAS) have identified >240 loci associated with type 2 diabetes (T2D), however most loci have been identified in analyses of European-ancestry individuals. To examine T2D risk in East Asian individuals, we meta-analyzed GWAS data in 77,418 cases and 356,122 controls. In the main analysis, we identified 298 distinct association signals at 178 loci, and across T2D association models with and without consideration of body mass index and sex, we identified 56 loci newly implicated in T2D predisposition. Common variants associated with T2D in both East Asian and European populations exhibited strongly correlated effect sizes. New associations include signals in/near GDAP1, PTF1A, SIX3, ALDH2, a microRNA cluster, and genes that affect muscle and adipose differentiation. At another locus, eQTLs at two overlapping T2D signals act through two genes, NKX6-3 and ANK1, in different tissues. Association studies in diverse populations identify additional loci and elucidate disease genes, biology, and pathways.\n\nType 2 diabetes (T2D) is a common metabolic disease primarily caused by insufficient insulin production and/or secretion by the pancreatic {beta} cells and insulin resistance in peripheral tissues1. Most genetic loci associated with T2D have been identified in populations of European (EUR) ancestry, including a recent meta-analysis of genome-wide association studies (GWAS) of nearly 900,000 individuals of European ancestry that identified >240 loci influencing the risk of T2D2. Differences in allele frequency between ancestries affect the power to detect associations within a population, particularly among variants rare or monomorphic in one population but more frequent in another3,4. Although smaller than studies in European populations, a recent T2D meta-analysis in almost 200,000 Japanese individuals identified 28 additional loci4. The relative contributions of different pathways to the pathophysiology of T2D may also differ between ancestry groups. For example, in East Asian (EAS) populations, T2D prevalence is greater than in European populations among people of similar body mass index (BMI) or waist circumference5. We performed the largest meta-analysis of East Asian individuals to identify new genetic associations and provide insight into T2D pathogenesis.

genetics↗