Biology subjects

Tan, K. W.

Publications and source records attributed to Tan, K. W..

13 recordsLinked to original sources

bioRxiv2026

Mitochondria-insulin granule crosstalk controls the early stages of granule maturation

Insulin secretion from pancreatic {beta}-cells is a tightly controlled process where hormone synthesis, granule formation and release are regulated in order to maintain whole body glucose homeostasis. Failure to produce or release insulin results in hyperglycemia that may develop into diabetes. Insulin-containing granules exist in different pools that have different propensity for release, yet what determined the fate of a granule after initial formation is not clear. In this study we aimed to identify key steps in the early life of an insulin granule that directs it towards release. Using two different methods for time-dependent labeling, we found that insulin granules shortly after budding from the trans-Golgi network associate with mitochondria. This organelle interaction involves the voltage-dependent anion channel (VDAC) and the vesicular nucleotide transporter (VNUT). Reduced VNUT expression prevented the recruitment of VDAC to insulin granules and redirected granules towards autophagy-dependent lysosomal degradation, resulting in reduced insulin content and impaired insulin secretion. These results show the requirement of granule-mitochondria crosstalk for normal progression through the early stages of the secretory pathway.

cell biology↗

bioRxiv2026

Isolation and Characterization of a Novel Calcium-Precipitating Alkali-Tolerant Lysinibacillus sphaericus in Local Urban Environment

Concrete is an extensively used construction material in infrastructures due to cost-effectiveness and durability. However, its low tensile strength makes it prone to cracking, posing persistent maintenance challenges. Traditional repair methods are often expensive and difficult to implement, especially for critical structures. Recent studies propose a potential solution involving alkaliphilic or alkali-tolerant calcium-carbonate producing bacteria in aiding concrete crack repair through microbial-induced calcium carbonate precipitation (MICP) via urea hydrolysis. Considering the adaptive nature of bacteria, we hypothesize that naturally occurring concrete-inhabiting microbes possess properties crucial for mending cracks. Therefore, this study aims to isolate and evaluate these inherent microbes and their MICP ability. Six concrete samples were collected and cultured on Alkaline Nutrient Agar for microbial isolation. Urease activity was assessed phenotypically and genotypically. MICP activity was observed and validated under Scanning Electron Microscope/Energy Dispersive Spectroscopy (SEM/EDS). Out of the 49 isolates cultured, Lysinibacillus sphaericus PUMA0250 emerges as the most promising isolate for practical implementation, showing high pH survivability in an in-house formulated concrete agar medium (pH 12.6). L. sphaericus PUMA0250 exhibited MICP activity via urea hydrolysis and harbored all crucial genes involved in urease-mediated MICP. SEM/EDS analysis confirmed the structure and composition of the produced calcium carbonate precipitate. This study demonstrates the potential of L. sphaericus PUMA0250 to be incorporated into concrete to assess its healing efficiency. Its ability to thrive in the alkaline concrete environment and produce calcium by-products may aid concrete crack repair, opening microbiological avenues for sustainable and efficient concrete repair methodologies. IMPORTANCEAlthough concrete is widely used in construction, the construction material is prone to cracking, leading to costly repairs and structural degradation. This study isolated Lysinibacillus sphaericus PUMA0250 from tropical urban concrete, that can survive the highly alkaline environment typical of concrete structures in equatorial climate. Importantly, it produces calcium carbonate, a mineral that can help in concrete repair. These findings contribute to our understanding of how microbes that naturally produce calcium carbonate by-products can be used to repair concrete. For such applications, microbes must be able to withstand extreme conditions, including high pH and low nutrient availability. Our discovery of a resilient, native isolate highlights the potential of tropical concrete microbiomes as sources for self-healing construction technologies. This work lays the foundation for more sustainable, low-maintenance building materials and opens new possibilities in microbiology-driven solutions for urban infrastructure challenges.

microbiology↗

bioRxiv2025

In silico discovery of pathogenic PD-L1 nsSNVs with altered glycosylation and immunotherapy binding

Immune checkpoint inhibitors (ICIs), particularly anti-PD-L1 monoclonal antibodies, block extracellular interactions between programmed death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) to enhance antitumour immunity. Here, we present a streamlined workflow integrating public databases, bioinformatics tools, and in silico molecular dynamics (MD) simulations and in vitro experimental assays to identify consequential pathogenic nonsynonymous PD-L1 variants (PD-L1SNVs). Four variants predicted as pathogenic across six bioinformatic tools (SIFT, PolyPhen-2, PANTHER, SNP-PhD, SNP&GO, and Pmut) destabilized the anti-PD-L1 ICI atezolizumab-drug binding epitope in MD studies, correlating with reduced drug affinity. Live-cell assays directly linked PD-L1SNVs to intracellular trafficking defects and aberrant glycosylation, even distal from N-glycosylation sites. Allostery modeling (AlloSigMA) linked these disruptions to long-range structural perturbations. Our work establishes bioinformatics-driven predictions of functionally impactful pathogenic PD-L1SNVs and highlight how glycosylation/trafficking defects may serve as key mechanisms compromising therapeutic efficacy. These insights in predicting and validating pathogenic PD-L1SNVs has significant implications for guiding the personalized selection of effective PD-L1-targeted therapies in clinical settings. One Sentence Summary: Uncovering the implication of non-synonymous single nucleotide variants (nsSNVs) on the structure and function of Programmed Death Ligand-1.

bioinformatics↗

bioRxiv2024

Cell Cycle Regulation has Shaped Budding Yeast Replication Origin Structure and Function

Eukaryotic DNA replication initiates from multiple genomic loci known as origins. At budding yeast origins like ARS1, a double hexamer (DH) of the MCM replicative helicase is assembled by Origin Recognition Complex (ORC), Cdc6 and Cdt1 via sequential hexamer loading from two opposed ORC binding sites. Cyclin Dependent Kinase (CDK) inhibits DH assembly, which prevents re-replication by restricting helicase loading to G1 phase. Here we show that an intrinsically disordered region (IDR) in the Orc2 subunit promotes interaction between ORC and the first loaded, closed-ring MCM hexamer (the MO intermediate); CDK phosphorylation of this IDR blocks MO formation and DH assembly. We show that MO functions by stabilising ORC at the lower affinity binding sites required for second hexamer loading. Origins comprising two high affinity ORC sites can assemble DH efficiently without MO by independently loading single hexamers; these origins escape CDK inhibition in vitro and in vivo. Our work reveals mechanistic plasticity in MCM loading with implications for understanding how CDK regulation has shaped yeast origin evolution and how natural origins might escape cell cycle regulation. We also identify key steps common to loading pathways, with implications for understanding how MCM is loaded in other eukaryotes.

biochemistry↗

bioRxiv2022

Efficacy of Non-invasive Brain Stimulation on Vision: A Systematic Review and Meta-analysis

ObjectiveMultiple studies have explored the use of non-invasive brain stimulation (NIBS) to enhance visual function. These studies vary in sample size, outcome measures, and NIBS methodology. We conducted a systematic review and meta-analyses to assess the effects of NIBS on visual functions in human participants with normal vision. MethodsWe followed the PRISMA guidelines, and a review protocol was registered with PROSPERO before study commencement (CRD42021255882). We searched Embase, Medline, PsychInfo, PubMed, OpenGrey and Web of Science using relevant keywords. The search covered the period from 1st January 2000 until 1st September 2021. Comprehensive meta-analysis (CMA) software was used for quantitative analysis. ResultsForty-nine studies were included, of which 19 were included in a meta-analysis (38.8%). Meta-analysis indicated acute (Hedgess g=0.232, 95% CI: 0.023-0.442, p=0.029) and aftereffects (0.590, 95% CI: 0.182-0.998, p=0.005) of transcranial electrical stimulation (tES, including three different stimulation protocols) on contrast sensitivity. Visual evoked potential (VEP) amplitudes were significantly enhanced immediately after tES (0.383, 95% CI: 0.110-0.665, p=0.006). Both tES (0.563, 95% CI: 0.230 to 0.896, p=0.001)] and anodal-transcranial direct current stimulation (a-tDCS) alone (0.655, 95% CI: 0.273 to 1.038, p=0.001) reduced crowding in peripheral vision. The effects of NIBS on visual acuity, motion perception and reaction time were not statistically significant. ConclusionsThere are significant effects of visual cortex NIBS on contrast sensitivity, VEP amplitude, an index of cortical excitability, and crowding among normally sighted individuals. Future studies with robust experimental designs are needed to substantiate these findings in populations with vision loss. PROSPERO registration numberCRD42021255882 HighlightsO_LIWe conducted a meta-analysis and a systematic review on the efficacy of non-invasive brain stimulation for improving on visual function C_LIO_LIVisual cortex non-invasive brain stimulation can enhance contrast sensitivity, reduce crowding in peripheral vision and enhance visually evoked potential amplitude among normally sighted individuals. C_LI

neuroscience↗

bioRxiv2022

ATPase activity of DFCP1 controls selective autophagy

Cellular homeostasis is governed by removal of damaged organelles and protein aggregates by selective autophagy mediated by cargo adaptors such as p62/SQSTM1. Autophagosomes can assemble in specialized cup-shaped regions of the endoplasmic reticulum (ER) known as omegasomes, which are characterized by the presence of the ER protein DFCP1/ZFYVE1. The function of DFCP1 is unknown, as are the mechanisms of omegasome formation and constriction. Here, we demonstrate that DFCP1 is an ATPase that dimerizes in an ATP-dependent fashion. Whereas depletion of DFCP1 had a minor effect on bulk autophagic flux, DFCP1 was required to maintain the autophagic flux of p62 under both fed and starved conditions, and this was dependent on its ability to bind and hydrolyse ATP. While DFCP1 mutants defective in ATP binding or hydrolysis localized to forming omegasomes, these omegasomes failed to constrict. Consequently, the release of nascent autophagosomes from omegasomes was markedly delayed. DFCP1 was found to associate with ubiquitinated proteins, and degradation of ubiquitinated cargoes such as protein aggregates, mitochondria and micronuclei was strongly inhibited when DFCP1 was knocked out or mutated. Thus, DFCP1 mediates ATPase-driven constriction of omegasomes to release autophagosomes for selective autophagy.

cell biology↗

bioRxiv2021

Dephosphorylation of the pre-initiation complex during S-phase is critical for origin firing

Genome stability requires complete DNA duplication exactly once before cell division. In eukaryotes, cyclin-dependent kinase (CDK) plays a dual role in this regulation by inhibiting helicase loading factors before also activating origin firing. CDK activates initiation by phosphorylation of two substrates, Sld2 and Sld3, forming a transient and limiting intermediate - the pre-initiation complex (pre-IC). The importance and mechanism of dissociation of the pre-IC from origins is not understood. Here we show in the budding yeast Saccharomyces cerevisiae that CDK phosphorylation of Sld3 and Sld2 is specifically and rapidly turned over during interphase by the PP2A and PP4 phosphatases. Inhibiting dephosphorylation of Sld3/Sld2 causes dramatic defects in replication initiation genome-wide, retention of the pre-IC at origins and cell death. These studies not only provide a mechanism to guarantee that Sld3 and Sld2 are dephosphorylated before helicase loading factors but also uncover a novel positive role for phosphatases in eukaryotic origin firing.

cell biology↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp3 Papain-like Protease

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom chemical library from which we identified Dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.

biochemistry↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

biochemistry↗

bioRxiv2021

Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order to identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play a key role in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.

bioengineering↗

bioRxiv2020

JIP4 is recruited by the phosphoinositide-binding protein Phafin2 to promote recycling tubules on macropinosomes

Macropinocytosis allows cells to take up extracellular material in a non-selective manner. The molecular mechanisms that mediate recycling of membranes and transmembrane proteins from macropinosomes still need to be defined. Here we report that JIP4, a coiled-coil containing protein previously described to bind to microtubule motors, is recruited to retromer- and actin-containing tubulating subdomains on macropinosomes by binding to the PH domain of the phosphatidylinositol 3-phosphate (PtdIns3P)-binding protein Phafin2. This recruitment is not shared by the closely related isoforms JIP3 and Phafin1. Disruption of Phafin2 or PtdIns3P impairs JIP4 recruitment to macropinosomes whereas forced localization of Phafin2 to mitochondria causes mitochondrial targeting of JIP4. While knockout of JIP4 suppresses tubulation, overexpression enhances tubulation from macropinosomes. JIP4 knockout cells display increased retention of macropinocytic cargo in both early and late macropinosomes, consistent with a recycling defect. Collectively, these data identify JIP4 and Phafin2 as components of a tubular recycling pathway that operates from macropinosomes.

cell biology↗

bioRxiv2017

Molecular basis for the maintenance of lipid asymmetry in the outer membrane of Escherichia coli

A distinctive feature of the Gram-negative bacterial cell envelope is the asymmetric outer membrane (OM), where lipopolysaccharides (LPS) and phospholipids (PLs) reside in the outer and inner leaflets, respectively. This unique lipid asymmetry renders the OM impermeable to external insults. In Escherichia coli, the OmpC-MlaA complex is believed to maintain lipid asymmetry by removing mislocalized PLs from the outer leaflet of the OM. How it performs this function is unknown. Here, we define the molecular architecture of the OmpC-MlaA complex to gain insights into its role in PL transport. We establish that MlaA sits entirely within the bilayer in complex with OmpC and provides a hydrophilic channel possibly for PL translocation across the OM. Furthermore, we show that flexibility in a hairpin loop adjacent to the channel modulates MlaA activity. Finally, we demonstrate that OmpC plays an active role in maintaining OM lipid asymmetry together with MlaA. Our work offers glimpses into how the OmpC-MlaA complex transports PLs across the OM and has important implications for future antimicrobial drug development.

biochemistry↗

bioRxiv2017

The PtdIns3P-binding protein Phafin2 escorts macropinosomes through the cortical actin cytoskeleton

Uptake of large volumes of extracellular fluid by actin-dependent macropinocytosis plays important roles in infection, immunity and cancer development. A key question is how large macropinosomes are able to squeeze through the dense actin network underlying the plasma membrane in order to move towards the cell centre for maturation. Here we show that, immediately after macropinosomes have been sealed off from the plasma membrane, the PH-and FYVE domain-containing protein Phafin2 is recruited by a mechanism that involves binding to phosphatidylinositol 3-phosphate (PtdIns3P) generated in a non-canonical manner. Phafin2 in turn regulates the actin cross-linking protein Filamin A to promote entry of macropinosomes through the subcortical actin matrix and subsequent maturation. Depletion of Phafin2 inhibits macropinocytic internalization and maturation. We conclude that PtdIns3P and its effector Phafin2 are key components of a system that allows nascent macropinosomes to navigate through the dense subcortical actin network.

cell biology↗