Biology subjects

Lai, S. K.

Publications and source records attributed to Lai, S. K..

13 recordsLinked to original sources

bioRxiv2025

A First-in-Class Therapy Targeting Transcription-Replication Conflicts Drives Synthetic Lethality in Homologous Recombination Deficient Cancers

Hidden beneath the layer of tumor malignancy lies unchecked proliferation driven by hyperactive transcription, which fosters the formation of RNA:DNA hybrids. These structural intermediates precipitate collisions between the transcription and replication machineries. Central to mitigating this genomic threat is interleukin enhancer-binding factor 2 (ILF2), which orchestrates the recruitment of RNA:DNA helicases to resolve R-loops and, in doing so, reveals cancer cells dependency on stress-mitigating mechanisms to avert genome catastrophe. Our discovery of Molephantin and its lead derivative, NYH0002 is the outcome of delineating ILF2s function. NYH002 exerts direct binding to the ILF2 complex and disrupts RNA:DNA helicase activity to elicit genome-wide DNA breakages. Tumors with elevated cyclin E and E2F1 expression are sensitive to NYH002, while those deficient in homologous recombination repair exhibit lethality. Collectively, these findings position ILF2 as a key regulator of R-loop homeostasis and reveal a therapeutic vulnerability that NYH002 exploits to suppress tumor malignancy.

cancer biology↗

bioRxiv2025

Omics analysis reveals striking effects of progesterone receptor on mitochondria and mitochondria-mediated apoptosis independent of caspases in Breast Cancer cells

The role of progesterone receptor (PR) in breast cancer remains controversial with conflicting reports from clinical and laboratory studies. To address these discrepancies, we conducted an integrated omics analysis of effects of agonist-activated PR in MCF-7 cells with elevated PR expression. PR agonist R5020 exerted strong antiproliferative and proapoptotic effects in these cells. Quantitative proteomics identified 4,915 PR-regulated proteins and 678 phosphorylated peptides, with nearly 100% verifiable rate by Western blotting analysis. The proteomics data was closely correlated with transcriptomic data. Key pathways upregulated included hypoxia, p53 signalling, TNFA signalling via NFKB, epithelial-mesenchymal transition, and KRAS signalling, while E2F targets, G2/M checkpoint, and mitotic spindle assembly were downregulated. R5020 broadly suppressed cell cycle regulators, including CDKs, cyclins, DNA replication proteins, and all components of the Ndc80 complex and chromosomal passenger complexes. Concurrently, it elicited significant changes in 200 mitochondrial proteins, upregulating many proapoptotic factors (e.g., BNIP3, NIX, AIF/AIFM1, AIFM2, ENDOG, HtrA2/Omi, Smac/DIABLO) and downregulating anti-apoptotic proteins (BCL-2, BCL-XL). This culminated in mitochondria-mediated apoptosis independent of effector caspases. The omics analysis also detected previously reported upregulation of pro-growth proteins such as EGFR, IRS2, and CCND1, but the upregulation was functionally futile and inhibitory phosphorylation of IRS2 at S306 increased 4-fold. In conclusion, this omics study achieved to date the most comprehensive and holistic understanding of PR-regulated proteins and molecular networks that are strongly anti-proliferative and proapoptotic with significant involvement of mitochondria. These findings suggest that pure PR agonists warrant evaluation as first-line endocrine therapy for breast cancer with high PR expression.

cancer biology↗

bioRxiv2024

A Droplet Digital PCR Assay for Quantification of Bacteriophage Viral Vector Titer and Purity.

PurposeBacteriophage (phage) based vectors offer considerable promise as tools for tuning the microbiome with molecular and genetic precision. However, standardized methods to rigorously characterize phage vectors remain lacking. Here, we present an optimized digital droplet PCR (ddPCR)-based assay for quantifying both the purity and potency of phage vector preparations. MethodsWe utilized central composite design to develop a ddPCR assay capable of quantifying the number of phage vector capsids packed with the phage vector genome or packed with the transgenic DNA of interest. This assay targets 2 unique DNA barcodes, designed to be biologically inert and maximally orthogonal to existing DNA sequences. ResultsThrough stringent optimization, we were able to achieve assay conditions that enable a dynamic range of nearly 3 orders of magnitude and correct for systemic error in the assay. We then show that biological activity assays consistently underestimate transgene-packed vectors titers, leading to overestimation of true transduction efficiency, particularly when contamination by genome-packed vectors is high. We further demonstrate how this approach facilitates optimization of vector production conditions and substantially improves the precision and reproducibility of phage vector transduction. ConclusionCompared to assays of biological activity, this optimized ddPCR assay has improved accuracy and, through design of experiments optimization, high precision (CVs = 5.5 {+/-} 1.3% and 4.5 {+/-} 1.0% for the genome and transgene barcodes, respectively). This assay can be broadly adopted to characterize and quality control vector preparations for various applications.

microbiology↗

bioRxiv2024

Evidence that the cell glycocalyx envelops respiratory syncytial virus (RSV) particles that form on the surface of RSV-infected human airway cells.

We examined how respiratory syncytial virus (RSV) particles circumvent the overlying glycocalyx on virus-infected A549 cells. The glycocalyx was detected using the lectin WGA-AL488 probe, and the antibodies anti-HS and anti-syndecan-4 that detect heparin sulphate (HS) and the syndecan-4 protein (SYND4) respectively. Imaging of RSV-infected cells provided evidence that the glycocalyx envelopes the virus filaments as they form, and that components of the glycocalyx such as HS moieties and SYND4 are displayed on the surface of the mature virus filaments. Using recombinant expression of the G protein we also demonstrated that the G protein was trafficked into pre-existing filamentous cellular structures with a well-defined glycocalyx, suggesting that the glycocalyx is maintained at the site of virus particle assembly. These data provide evidence that during RSV particle assembly the virus filaments become enveloped by the glycocalyx, and that the glycocalyx should be considered as a structural component of RSV particles.

microbiology↗

bioRxiv2024

Evidence that the SARS-CoV-2 S protein undergoes a conformational change at the Golgi Complex that leads to the formation of virus neutralising antibody binding epitopes in the S1 protein subunit.

Recombinant SARS-CoV-2 S protein expression was examined in Vero cells by imaging using the human monoclonal antibody panel (PD4, PD5, sc23, and sc29). The PD4 and sc29 antibodies recognised conformational specific epitopes in the S2 protein subunit at the Endoplasmic reticulum and Golgi complex. While PD5 and sc23 detected conformationally specific epitopes in the S1 protein subunit at the Golgi complex, only PD5 recognised the receptor binding domain (RBD). A comparison of the staining patterns of PD5 with non-conformationally specific antibodies that recognises the S1 subunit and RBD suggested the PD5 recognised a conformational structure within the S1 protein subunit. Our data suggests the antibody binding epitopes recognised by the human monoclonal antibodies formed at different locations in the secretory pathway during S protein transport, but a conformational change in the S1 protein subunit at the Golgi complex formed antibody binding epitopes that are recognised by virus neutralizing antibodies.

microbiology↗

bioRxiv2022

Erythrocyte signalling is critical for Plasmodium falciparum invasion

Successful Plasmodium falciparum merozoite invasion requires the activation of red blood cell (RBC) signalling pathways. The binding of parasite ligand reticulocyte binding protein homologue 5 (RH5) to its host receptor Basigin is essential for merozoite invasion and triggers a Ca2+ influx in RBCs. Here we observed that RH5-bound RBCs form a multimeric protein complex containing Basigin, CD44 and {beta}2-adrenergic receptor ({beta}2AR), suggesting that RH5-Basigin interaction is functionally associated with the host cAMP signalling pathway. Interestingly, we detected a characteristic rise in cAMP levels in the RBC upon RH5-Basigin interaction, which can be blocked by G protein and cAMP-synthesising adenylyl cyclase (AC) inhibitors. Furthermore, we demonstrated that RBC L-type Ca2+ channel inhibitor and cAMP signalling inhibitors are able to block merozoite invasion. Checkerboard invasion inhibition assay containing different combinations of signalling inhibitors also exhibited a drastic amplification of inhibition levels, indicating that these signalling proteins are functioning in a common signalling cascade to activate the L-type Ca2+ channels. Taken together, this study provides new insights into the role of a host cAMP-Ca2+ signalling pathway during merozoite invasion and sheds new light on antimalarial therapeutic strategies to tackle the high infection rate and growing threat of drug resistant parasites. Key PointsO_LIA pre-existing Basigin-associated membrane protein complex undergoes increased protein assembly upon RH5 binding on the RBC surface. C_LIO_LIPlasmodium falciparum merozoite exploits host cAMP signalling to initiate Ca2+ influx in the RBC. C_LI

microbiology↗

bioRxiv2022

Genomic, transcriptomic, and metabolomic analysis of Traditional Chinese Medicine plant Oldenlandia corymbosa reveals the biosynthesis and mode of action of anti-cancer metabolites

Natural products from traditional medicinal plants are valuable candidates for clinical cancer therapy. Plants from the Oldenlandia-Hedyotis complex are popular ingredients of Traditional Chinese Medicine (TCM), however a major hurdle in the plant bioprospecting process of TCM plants is that the active metabolites, their biosynthetic pathways, and mode of action are often unknown. We show that Oldenlandia corymbosa extracts are active against breast cancer cell lines. To study the genes involved in the biosynthesis of active compounds in this medicinal plant, we assembled a high-quality genome. We show that the main active compound is ursolic acid and that abiotic stresses cause changes in anti-cancer activity, metabolite composition, and gene expression of plants. To reveal the mode of action of ursolic acid, we show that cancer cells undergo mitotic catastrophe, and we identify three high-confidence protein binding targets by Cellular Thermal Shift Assay (CETSA) and reverse docking.

plant biology↗

bioRxiv2022

Stable nebulization and muco-trapping properties of Regdanvimab/IN-006 support its development as a potent, dose-saving inhaled therapy for COVID-19

The respiratory tract represents the key target for antiviral delivery in early interventions to prevent severe COVID-19. While neutralizing monoclonal antibodies (mAb) possess considerable efficacy, their current reliance on parenteral dosing necessitates very large doses and places a substantial burden on the healthcare system. In contrast, direct inhaled delivery of mAb therapeutics offers the convenience of self-dosing at home, as well as much more efficient mAb delivery to the respiratory tract. Here, building on our previous discovery of Fc-mucin interactions crosslinking viruses to mucins, we showed that regdanvimab, a potent neutralizing mAb already approved for COVID-19 in several countries around the world, can effectively trap SARS-CoV-2 virus-like-particles in fresh human airway mucus. IN-006, a reformulation of Regdanvimab, was stably nebulized across a wide range of concentrations, with no loss of activity and no formation of aggregates. Finally, nebulized delivery of IN-006 resulted in 100-fold greater mAb levels in the lungs of rats compared to serum, in marked contrast to intravenously dosed mAbs. These results not only support our current efforts to evaluate the safety and efficacy of IN-006 in clinical trials, but more broadly substantiate nebulized delivery of human antiviral mAbs as a new paradigm in treating SARS-CoV-2 and other respiratory pathologies.

bioengineering↗

bioRxiv2022

Modeling predicts mechanisms altered by mutations of the SARS-CoV-2 delta and omicron variants

We apply our mechanistic, within-host, pre-immunity, respiratory tract infection model for unvaccinated, previously uninfected, and immune-compromised individuals. Starting from published cell infection and viral replication data for the SARS-CoV-2 alpha variant, we explore variability in outcomes of viral load and cell infection due to three plausible mechanisms altered by SARS-CoV-2 mutations of delta and omicron. We seek a mechanistic explanation of clinical test results: delta nasal infections express [~]3 orders-of-magnitude higher viral load than alpha, while omicron infections express an additional 1 to 2 orders-of-magnitude rise over delta. Model simulations reveal shortening of the eclipse phase (the time between cellular uptake of the virus and onset of infectious viral replication and shedding) alone can generate 3-5 orders-of-magnitude higher viral load within 2 days post initial infection. Higher viral replication rates by an infected cell can generate at most one order-of-magnitude rise in viral load, whereas higher cell infectability has minimal impact and lowers the viral load.

immunology↗

bioRxiv2021

The cellular characterisation of SARS-CoV-2 spike protein in virus-infected cells using Receptor Binding Domain-binding specific human monoclonal antibodies.

A human monoclonal antibody panel (PD4, PD5, PD7, SC23 and SC29) was isolated from the B cells of convalescent patients and used to examine the S protein in SARS-CoV-2- infected cells. While all five antibodies bound conformational-specific epitopes within SARS-CoV-2 Spike (S) protein, only PD5, PD7, and SC23 were able to bind to the Receptor Binding Domain (RBD). Immunofluorescence microscopy was used to examine the S protein RBD in cells infected with the Singapore isolates SARS-CoV-2/0334 and SARS-CoV-2/1302. The RBD-binders exhibited a distinct cytoplasmic staining pattern that was primarily localised within the Golgi complex and was distinct from the diffuse cytoplasmic staining pattern exhibited by the non-RBD binders (PD4 and SC29). These data indicated that the S protein adopted a conformation in the Golgi complex that enabled the RBD recognition by the RBD-binders. The RBD-binders also recognised the uncleaved S protein indicating that S protein cleavage was not required for RBD recognition. Electron microscopy indicated high levels of cell-associated virus particles, and multiple cycle virus infection using RBD-binder staining provided evidence for direct cell-to-cell transmission for both isolates. Although similar levels of RBD-binder staining was demonstrated for each isolate, the SARS-CoV-2/1302 exhibited slower rates of cell-to-cell transmission. These data suggest that a conformational change in the S protein occurs during its transit through the Golgi complex that enables RBD recognition by the RBD-binders, and suggests that these antibodies can be used to monitor S protein RBD formation during the early stages of infection. ImportanceThe SARS CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection and the S protein RBD is therefore a focus of therapeutic and vaccine interventions. However, new virus variants have emerged with altered biological properties in the RBD that can potentially negate these interventions. Therefore an improved understanding of the biological properties of the RBD in virus-infected cells may offer future therapeutic strategies to mitigate SARS CoV-2 infection. We used physiologically relevant antibodies that were isolated from the B cells of convalescent COVID19 patients to monitor the RBD in cells infected with SARS CoV-2 clinical isolates. These immunological reagents specifically recognise the correctly folded RBD and were used to monitor the appearance of the RBD in SARS CoV-2-infected cells and identified the site where the RDB first appears.

microbiology↗

bioRxiv2021

Hexavalent Sperm-Binding IgG Antibody Released from Self-Dissolving Vaginal Film Enables Potent, On-Demand Non-Hormonal Female Contraception

Non-hormonal products for on-demand contraception are a global health technology gap, motivating us to pursue the use of sperm-binding monoclonal antibodies as a strategy to enable safe, effective, desirable, on-demand contraception. Here, using cGMP-compliant Nicotiana-expression system, we produce an ultra-potent sperm-binding IgG antibody possessing 6 Fab arms per molecule that bind a well-established contraceptive antigen target, CD52g. We term this hexavalent antibody "Fab-IgG-Fab" (FIF) to reflect its molecular orientation. The Nicotiana-produced FIF exhibits at least 10-fold greater sperm agglutination potency and kinetics than the parent IgG, while preserving Fc-mediated trapping of individual spermatozoa in mucus. We formulate the Nicotiana-produced FIF into a polyvinyl alcohol-based water-soluble contraceptive film, and evaluate its potency in reducing progressively motile sperm in the sheep vagina. Two minutes after vaginal instillation of human semen, no progressively motile sperm are recovered from the vaginas of sheep receiving FIF-Film. In contrast, high numbers of progressively motile sperm are recovered from sheep receiving a placebo film control. Our work supports the potential of highly multivalent contraceptive antibodies to provide safe, effective, on-demand non-hormonal contraception.

bioengineering↗

bioRxiv2020

Plasmodium falciparum Replication factor C subunit 1 is involved in genotoxic stress response

About half the worlds population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin. The replication factor C subunit 1 protein is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localization to the nuclear periphery indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions. Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication and DNA repair pathways leading to cell death. Additionally, mislocalization of the endogenously tagged protein confirmed its essential role in parasites replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in plasmodium falciparum. ImportanceFrontline drugs have been found to induce DNA damage in the human malaria parasite Plasmodium falciparum. The genotoxic stress response in Plasmodium and the interplay between DNA damage repair, replication and activation of programmed cell death pathways remains largely undescribed. This study shows a distinct pattern of localization of PfRFC1 during replication and DNA repair. PfRFC1 responds to genotoxic stress with an increase in protein expression. Interfering with the RFC complex formation or mislocalization of PfRFC1 is associated with disrupted genotoxic stress response. Additionally, a reduction of PfRFC1 protein levels is observed upon treatment with antimalarial drugs or under apoptosis like conditions, highlighting the role of DEVD/G like motif in mediating programmed cell death in these parasites. This study sheds light on the role of PfRFC1 in differentially responding to replication, genotoxic stress and programmed cell death in Plasmodium parasites.

cell biology↗

bioRxiv2020

Engineering highly multivalent sperm-binding IgG antibodies for potent non-hormonal female contraception

Many women risk unintended pregnancy due to dissatisfaction with available hormonal contraceptive methods. This led us to pursue topical sperm-binding monoclonal antibodies as a strategy for safe, non-hormonal contraception. Motivated by the greater agglutination potencies of polymeric immunoglobulins such as IgM and the exceptional bioprocessing ease in manufacturing IgG, we engineered IgGs possessing 6-10 Fabs against a unique surface antigen universally present on human sperm. These highly multivalent IgGs (HM-IgGs) are at least 10- to 16-fold more potent and faster than the parent IgG at agglutinating sperm, while preserving Fc-mediated trapping of individual spermatozoa in mucus. The increased potencies translate to effective (>99.9%) reduction of progressively motile sperm in the sheep vagina using 33 micrograms of the 10 Fab HM-IgG. HM-IgGs produce at comparable yields and possess identical thermal stability to the parent IgG, with greater homogeneity. HM-IgGs represent not only promising biologics for non-hormonal contraception but also a promising platform for generating potent agglutinating mAb for diverse medical applications.

molecular biology↗