Biology subjects

Yap, J.

Publications and source records attributed to Yap, J..

9 recordsLinked to original sources

bioRxiv2025

Single cell functional immunogenomics of the fallopian tube reveals a precursor immune surveillance network for ovarian cancer prevention

The fallopian tube (FT) is increasingly recognized as the site of origin for high-grade serous ovarian cancer (HGSOC), yet its immune landscape and potential role in tumor surveillance remain poorly understood. Here, we employ single-cell RNA sequencing (scRNA-seq) and paired single-cell T-cell receptor sequencing (scTCR-seq) to profile tissue-resident memory-like T cells (TRMLs) from matched non- cancerous FT, metastatic omental tumors, and peripheral blood in HGSOC patients. Surprisingly, we identify a substantial clonal and functional overlap between TRMLs from non-cancerous FT and tumor- infiltrating TRMLs, with 18.4% of tumor TRML clonotypes being shared with FT TRMLs, significantly exceeding overlap with circulating T cells. Shared TCR clonotypes are preferentially enriched in exhausted CD8+ T cells (CD8-Tex) and proliferative exhausted CD8+ subsets, suggesting prior antigenic exposure. Notably, we identify a previously uncharacterized CD8+ SIK3-high subset enriched in tumors, with gene expression signatures implicating epigenetic plasticity and metabolic adaptation. Functionally, non-cancerous FT-derived TRMLs recognize autologous tumor antigens, as evidenced by robust IFN-{gamma} responses in tumor organoid co-culture assays and CD137 upregulation. Whole-genome and transcriptomic analyses reveal shared somatic mutations between the FT and tumor, supporting the FT as the tumors evolutionary origin. Predicted tumor neoantigens elicit TRML responses in the non-cancerous FT, and single-cell TCR profiling confirms that neoantigen-reactive T cells in FT share clonotypes with tumor-infiltrating TRMLs, further reinforcing their role in early immune surveillance. Importantly, FT-derived TRMLs exhibit lower exhaustion signatures than their tumor counterparts, suggesting their potential as a preferable source for adoptive T cell therapies. Our findings uncover a precursor immune surveillance network in the FT and provide a rationale for leveraging FT-resident T cells in cancer immunotherapy and prevention.

immunology↗

bioRxiv2024

Spatiotemporal control of subcellular O-GlcNAc signaling using Opto-OGT

The posttranslational modification of intracellular proteins through O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is a conserved regulatory mechanism in multicellular organisms. Catalyzed by O-GlcNAc transferase (OGT), this dynamic modification plays an essential role in signal transduction, gene expression, organelle function, and systemic physiology. Here we present Opto-OGT, an optogenetic probe that allows for precise spatiotemporal control of OGT activity through light stimulation. By fusing a photosensitive cryptochrome protein to OGT, Opto-OGT can be robustly and reversibly activated with high temporal resolution by blue light and exhibits minimal background activity without illumination. Transient activation of Opto-OGT results in mTORC activation and AMPK suppression which recapitulate nutrient-sensing signaling. Furthermore, Opto-OGT can be customized to be localized at specific subcellular sites. By targeting OGT to the plasma membrane, we demonstrate downregulation of site-specific AKT phosphorylation and signaling outputs in response to insulin stimulation. Thus, Opto-OGT is a powerful tool to define the role of O-GlcNAcylation in cell signaling and physiology.

cell biology↗

bioRxiv2023

The staphylococcal type VII secretion system impacts daptomycin sensitivity through controlling bacterial cell envelope integrity

The human pathogen Staphylococcus aureus encodes a specialised type VII secretion system (T7SS), which plays an important role in bacterial virulence during infection. However, the functions the T7SS during infection and in bacterial physiology remain unclear. Here we demonstrate that S. aureus strains lacking the the T7SS effector EsxC ({Delta}esxC) was highly sensitive to the important last resort drug, daptomycin, as well as other membrane-targeting antibiotics, including gramicidin and bithionol. To understand how EsxC mediates increased antibiotic sensitivity, we investigated its functions in the staphylococcal cell envelope. Scanning electron microscopy analysis of an esxC mutant revealed a distinct cell surface morphology. Interestingly, {Delta}esxC displayed a decrease in membrane fluidity, altered membrane protein profiles and altered cell wall synthesis. The esxC mutant demonstrated enhanced daptomycin binding which correlated with the increased negative charge of mutant membranes. Calcium ions, which can bind membranes affecting charge, impacted growth of {Delta}esxC and sensitivity to daptomycin, suggesting that EsxC may modulate calcium binding to membranes. Furthermore, the esxC mutant displayed a heightened susceptibility to daptomycin during intracellular infection, and in a murine skin infection model. Thus, our data show that the T7SS effector EsxC impacts sensitivity of S. aureus to membrane-acting drugs such as daptomycin through modulation of cell membrane integrity, indicating its potential as a drug target. Author SummaryT7SS has a range of functions in bacteria including specific roles in bacterial physiology including DNA uptake, membrane integrity and bacterial development. In S. aureus T7SS has been shown to be critical for bacterial virulence, intra-species competition and in host cell interactions, although their functions in bacterial physiology are not clear. Here we report a role of the staphylococcal T7SS effector EsxC in the modulation of the cell membrane and surface integrity, which impacts the activity of membrane targeting drugs like daptomycin. Our data indicate that targeting this system could potentially enhance activity of existing therapeutic agents.

microbiology↗

bioRxiv2022

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

microbiology↗

bioRxiv2019

Development and evaluation of a rapid on-site water pathogen detection system for water quality monitoring

Timely response to outbreak of water-borne diseases caused by bacteria requires efficient monitoring and rapid detection strategies. Herein, we report a rapid DNA-based on-site detection system for specific detection of Pseudomonas aeruginosa. To evaluate the test performance of our method against spiked water samples, parallel tests based on real-time PCR and standard culture methods were concurrently performed. Test sensitivities of between 96.7% and 92.3% were obtained, based on the calculation obtained from qPCR and culture test, respectively, with a corresponding level of specificity of 92.9% and 83.3%. Time-to-result is around 45 min, with a detection limit of 1 CFU/100 mL. Here, a fully-deployable detection method where bacteria of-interest can be detected rapidly with high accuracy was described. This test method can be modified to detect other bacteria of-interest and can also be used in different applications. The test results can be obtained on-site and can therefore be particularly useful in public facilities health surveillance, where regulators can quickly determine if a site is safe or if other emergency response measures are required.\n\nIMPORTANCEPseudomonas aeruginosa is a well-established water quality biomarker, known to be associated to humans health risks. To ascertain the presence of this pathogen, relevant stakeholders currently rely on standard quantitative ISO methods (APHA 9213E) which require 6 days from sampling to results. This window could potentially lead to waterborne outbreaks if the contaminated water features are not shut down for proper and urgent mitigation. This manuscript describes a method to detect this disease-causing microorganism in its viable forms under 1 hour, with a sensitivity of 1 CFU/100mL. Besides providing valuable information of the quality of water system, this direct monitoring of pathogens can reduce substantial time needed from sampling to reporting. This method can be established as a platform technology for other pathogenic microorganisms. On-going work to develop economic point-of-care prototypes could facilitate quick screening of targeted waterborne pathogens and results in better assessment of public health risk and quick in devising emergency response measures and other management strategies.

microbiology↗

bioRxiv2019

Digital phenotyping by consumer wearables identifies sleep-associated markers of cardiovascular disease risk and biological aging

Despite growing adoption of consumer wearables, the potential for sleep metrics from these devices to contribute to sleep-related biomedical research remains largely uncharacterized. Here we analyze sleep tracking data, along with questionnaire responses and multi-modal phenotypic data, generated from 482 normal volunteers. First, we provide a detailed comparison of wearable-derived and self-reported sleep metrics, particularly total sleep time (TST) and sleep efficiency (SE). We then identified demographic, socioeconomic and lifestyle factors associated with wearable-derived sleep duration. We also analyzed our multi-modal phenotypic data and showed that wearable-derived TST and SE are associated with various cardiovascular disease risk markers, whereas self-reported measures were not. Using whole-genome sequencing data, we estimated leukocyte telomere length and showed that volunteers with insufficient sleep also exhibit premature telomere attrition. Our study highlights the potential for sleep metrics generated by consumer wearables to provide novel insights into data generated from population cohort studies.

epidemiology↗

bioRxiv2018

The Dimer-dependent Catalytic Activity of RAF Family Kinases Is Revealed Through Characterizing Their Oncogenic Mutants

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the {beta}3-C loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

cancer biology↗

bioRxiv2018

AMPKi overcomes the paradoxical activation of CRAF driven by RAF inhibitors through blocking the 14-3-3 binding to its carboxyl-terminus

The paradoxical activation of RAF kinase is the predominant challenge in cancer therapies with RAF inhibitors. The inhibitor-bound RAF molecules are able to transactivate their wild-type binding partners. 14-3-3 that binds to the carboxyl-terminus of RAF kinase has been suggested to regulate the dimer-dependent activation of RAF kinase under physiological conditions, though the molecular basis is not clear. In this study, we investigated the role of 14-3-3 in the paradoxical effect of RAF inhibitors. Firstly, we found that the 14-3-3 binding to the carboxyl-terminus of CRAF was essential for its transactivation. Further, we demonstrated that this binding enhanced the dimer affinity of CRAF. Since 14-3-3 binds to the phosphorylated motif, we next investigated and identified AMPK and CRAF itself as two putative kinases that phosphorylate redundantly the 14-3-3 binding motif of CRAF. Among RAF isoforms, CRAF plays a dominant role in the paradoxical effect of RAF inhibitors, and we thus determined whether the combinatory inhibition of AMPK and CRAF would block this effect. Indeed, our data showed that AMPKi not only blocked the RAF inhibitor-driven paradoxical activation of RAF signaling and cellular overgrowth in Ras-mutated cancer cells but also reduced the drug-resistant clones derived from BRAF(V600E)-mutated cancer cells. Finally, we showed that the 14-3-3 binding to the carboxyl-terminus of CRAF was dispensable for its catalytic function in vivo. Together, our study unraveled how 14-3-3 regulates the dimerization-driven RAF activation and identified AMPKi as a potential method to relieve the drug resistance and side effect of RAF inhibitors in cancer therapy.

cancer biology↗

bioRxiv2017

The Activation of MEK1 by Enhanced Homodimerization Drives Tumorigenesis

Hyperactive RAS/RAF/MEK/ERK signaling has a well-defined role in cancer biology. Aberrant pathway activation occurs mostly upstream of MEK; however, MEK mutations are prevalent in some cancer subsets. Here we show that cancer-related MEK mutants can be classified as those activated by relieving the inhibitory role of helix A, and those with in-frame deletions of {beta}3-C loop, which exhibit differential resistance to MEK inhibitors in vitro and in vivo. The {beta}3-C loop deletions activate MEK1 through enhancing homodimerization that can drive intradimer cross-phosphorylation of activation loop. Further, we demonstrate that MEK1 dimerization is required both for its activation by RAF and for its catalytic activity towards ERK. Our study identifies a novel group of MEK mutants, illustrates some key steps in RAF/MEK/ERK activation, and has important implications for the design of therapies targeting hyperactive RAS/RAF/MEK/ERK signaling in cancers.

cancer biology↗