Biology subjects

Tan, W. S.

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

6 recordsLinked to original sources

bioRxiv2025

Simulation-Guided Exploration of PAINT Parameter Space for Accurate Molecular Quantification

Molecular quantification using Point Accumulation for Imaging in Nanoscale Topography (PAINT) depends critically on probe kinetics, imaging conditions, and surface molecular properties. This study presents a simulation-guided framework to systematically explore the PAINT parameter space and identify conditions that enable molecular quantification with high accuracy. Detection thresholds for key PAINT outputs, namely point spread function density, localization cloud density, and binding event density, are defined to ensure [≥] 90% accuracy in density estimates and statistical interpretability of spatial distributions. A neural network surrogate model, trained on Monte Carlo simulations, is used to perform Sobol sensitivity analysis, revealing that probe kinetics and concentration are the dominant contributors to output variability. The model also enables rapid mapping of viable parameter regimes and shows that interpretable quantification of spatial distributions in high-density, clustered systems requires either a priori knowledge of the molecular architecture or improved spatial resolution. Overall, this framework provides quantitative guidance for optimizing PAINT experiments and supports the rational design of non-DNA-based, PAINT-compatible probes, thereby expanding the applicability of PAINT to a broader range of molecular systems.

biophysics↗

bioRxiv2020

Host cell factors important for BHV-1 cell entry revealed by genome-wide CRISPR knockout screen

In order to identify host factors that impact Bovine Herpes Virus Type 1 (BHV-1) infection we previously applied a genome wide CRISPR knockout screen with a library covering all bovine protein coding genes. We compiled a list of both pro-viral and anti-viral proteins involved in BHV-1 replication; here we provide further analysis of those that are potentially involved in viral entry into the host cell. These entry related factors include the cell surface proteins PVR and PVRL2, a group of enzymes directly or indirectly associated with the biosynthesis of Heparan Sulfate Proteoglycans (HSPG), and proteins that reside in the Golgi apparatus engaging in intra-Golgi trafficking. For the first time, we provide evidence that PVRL2 serves a receptor for BHV-1, mediating more efficient entry than the previously identified PVR. By knocking out two enzymes that catalyze HSPG chain elongation, HST2ST1 and GLCE, we demonstrated the significance of HSPG in BHV-1 entry. Another intriguing cluster of genes, COG1, COG2 and COG4-7 encodes for six subunits of the conserved oligomeric Golgi (COG) complex. MDBK cells lacking COG6 were less infectable by BHV-1 but release newly produced virions more efficiently as evidenced by fewer but bigger plaques compared to control cells, suggesting impaired HSPG biosynthesis. To facilitate candidate validation, we devised a one-step multiplex CRISPR interference (CRISPRi) system named CRISPR3i that enables quick and simultaneous deployment of three CRISPRs for efficient gene inactivation. Using CRISPR3i, we verified an additional 23 candidates, with many implicated in cellular entry.

microbiology↗

bioRxiv2020

Genome-wide CRISPR knockout screen reveals membrane tethering complexes EARP and GARP important for Bovine Herpes Virus Type 1 replication

We produced a genome wide CRISPR knockout library, btCRISPRko.v1, targeting all protein coding genes in the cattle genome and used it to identify host genes important for Bovine Herpes Virus Type 1 (BHV-1) replication. By infecting library transduced MDBK cells with a GFP tagged BHV-1 virus and FACS sorting them based on their GFP intensity, we identified a list of pro-viral and anti-viral candidate host genes that might affect various aspects of the virus biology, such as cell entry, RNA transcription and viral protein trafficking. Among them were VPS51, VPS52 and VPS53 that encode for subunits of two membrane tethering complexes EARP and GARP. Simultaneous loss of both complexes in MDBKs resulted in a significant reduction in the production of infectious cell free BHV-1 virions, suggesting the vital roles they play during capsid re-envelopment with endocytosed membrane tubules prior to endosomal recycling mediated cellular egress. We also observed potential capsid retention and aggregation in the nuclei of these cells, indicating that they might also indirectly affect capsid egress from the nucleus. The btCRISPRko.v1 library generated here greatly expanded our capability in BHV-1 related host gene discovery; we hope it will facilitate efforts intended to study interactions between the host and other pathogens in cattle and also basic host cell biology.

microbiology↗

bioRxiv2018

Structure of the Macrobrachium rosenbergii Nodavirus: A new genus within the Nodaviridae?

Macrobrachium rosenbergii nodavirus (MrNV) is a pathogen of freshwater prawns that poses a threat to food-security and causes significant economic losses in the aquaculture industries of many developing nations. A detailed understanding of the MrNV virion structure will inform the development of strategies to control outbreaks. The MrNV capsid has also been engineered to display heterologous antigens, thus knowledge of its atomic resolution structure will benefit efforts to develop tools based on this platform. Here we present an atomic-resolution model of the MrNV capsid protein, calculated by cryogenic electron microscopy (cryoEM) of MrNV virus-like particles (VLPs) produced in insect cells, and three-dimensional image reconstruction at 3.3 [A] resolution. CryoEM of MrNV virions purified from infected freshwater prawn post-larvae yielded a 6.6 [A] resolution structure confirming the biological relevance of the VLP structure.\n\nOur data revealed that unlike other known nodaviruses structures, which have been shown to assemble capsids having trimeric spikes, MrNV assembles a T=3 capsid with dimeric spikes. We also found a number of surprising similarities between the MrNV capsid structure and that of the Tombusviridae. 1. An extensive network of N-terminal arms lines the capsid interior forming long-range interactions to lace together asymmetric units. 2. The capsid shell is stabilised by three pairs of Ca2+ ions in each asymmetric unit. 3. The protruding spike domain exhibits a very similar fold to that seen in the spikes of the tombusviruses. These structural similarities raise questions concerning the correct taxonomic classification of MrNV.

microbiology↗

bioRxiv2017

A Low-Cost Multiplex Biomarker Assay Stratifies Colorectal Cancer Patient Samples into Clinically-Relevant Subtypes

Previously, we classified colorectal cancers (CRCs) into five CRCA subtypes with different prognoses and potential treatment responses, using a 786-gene signature. We merged our subtypes and those described by five other groups into four consensus molecular subtypes (CMS) that are similar to CRCA subtypes. Here we demonstrate the analytical development and application of a custom NanoString platform-based biomarker assay to stratify CRC into subtypes. To reduce costs, we switched from the standard protocol to a custom modified protocol (NanoCRCA) with a high Pearson correlation coefficient (>0.88) between protocols. Technical replicates were highly correlated (>0.96). The assay included a reduced robust 38-gene panel from the 786-gene signature that was selected using an in-laboratory developed computational pipeline of class prediction methods. We applied our NanoCRCA assay to untreated CRCs including fresh-frozen and formalin-fixed paraffin-embedded (FFPE) samples (n=81) with matched microarray or RNA-Seq profiles. We further compared the assay results with CMS classification, different platforms (microarrays/RNA-Seq) and gene-set classifiers (38 and 786 genes). NanoCRCA classified fresh-frozen samples (n=39; not including those showing a mixture of subtypes) into all five CRCA subtypes with overall high concordance across platforms (89.7%) and with CMS subtypes (84.6%), independent of tumour cellularity. This analytical validation of the assay shows the association of subtypes with their known molecular, mutational and clinical characteristics. Overall, our modified NanoCRCA assay with further clinical assessment may facilitate prospective validation of CRC subtypes in clinical trials and beyond.\n\nNovelty and ImpactWe previously identified five gene expression-based CRC subtypes with prognostic and potential predictive differences using a 786-gene signature and microarray platform. Subtype-driven clinical trials require a validated assay suitable for routine clinical use. This study demonstrates, for the first time, how molecular CRCA subtype can be detected using NanoString Technology-based biomarker assay (NanoCRCA) suitable for clinical validation. NanoCRCA is suitable for analysing FFPE samples, and this assay may facilitate patient stratification within clinical trials.

cancer biology↗

bioRxiv2017

Cryo-Electron Microscopy Structure of the Macrobrachium Rosenbergii Nodavirus Capsid at 7 Angstroms Resolution

White tail disease in the giant freshwater prawn Macrobrachium rosenbergii causes significant economic losses in shrimp farms and hatcheries and poses a threat to food-security in many developing countries. Outbreaks of Macrobrachium rosenbergii nodavirus (MrNV), the causative agent of white tail disease (WTD) are associated with up to 100% mortality rates. Recombinant expression of the capsid protein of MrNV in insect cells leads to the production of VLPs closely resembling the native virus. We have investigated the structure of MrNV VLPs by cryogenic electron microscopy, determining a structure of the viral capsid at 7 angstroms resolution. Our data show that MrNV VLPs package nucleic acids in a manner reminiscent of other known nodavirus structures. The structure of the capsid however shows striking differences from insect and fish infecting nodaviruses, which have been shown to assemble trimer-clustered T=3 icosahedral virus particles. MrNV particles have pronounced dimeric blade-shaped spikes extending up to 6 nm from the outer surface of the capsid shell. Our structural analysis supports the assertion that MrNV along with the related virus of marine shrimp Penaeus vannamei nodavirus (PvNV) may represent a new genus of the Nodaviridae.\n\nAuthor summaryMacrobrachium rosenbergii nodavirus (MrNV) is the causative agent of white tail disease (WTD) which leads to 100% mortality in shrimp-farms growing giant freshwater prawn (M. rosenbergii). MrNV is therefore a significant threat to food security and causes severe economic losses in developing countries such as Malaysia, Indonesia, Pakistan, Thailand and India. Here we have used electron microscopy to study the three-dimensional structure of MrNV, revealing that the viral capsid - the protein shell that encloses the viral genome, protecting it and transporting it from one host to the next - is differently organised to capsids produced by other viruses in the nodavirus family. The virus was found to have large blade-like spikes on its outer surface that are likely important in the early stages of infection, when the virus attaches to and enters a host cell.

microbiology↗