Biology subjects

Lai, E.

Publications and source records attributed to Lai, E..

8 recordsLinked to original sources

bioRxiv2026

Nanoscale 3D profiling of the T cell membrane reveals CD2 enrichment at microvilli tips, positioning adhesion near TCR zones in the immunological synapse

The T cell membrane features a specialized molecular and topological organization critical for signaling and immune function. During antigen detection, T cells utilize finger-like protrusions called microvilli to dynamically scan the antigen-presenting cell (APC) surface. Enriched with T cell receptors (TCRs) and key signaling molecules, microvilli serve as primary signaling hubs, yet their nanoscale architecture remains poorly defined. Upon antigen engagement, TCR activation drives the formation of the immunological synapse (IS), a highly organized membrane contact with the APC critical for T cell function. However, profiling IS architecture at the nanoscale remains technically challenging. Here, we introduce NanoMAP (Nanoscale Membrane Architecture Profiling), an expansion microscopy-based platform that resolves receptor organization on T cell microvilli and within the IS at 35-60 nm resolution. Using NanoMAP, we show that effector CD8+ T cells possess a 5.1-fold higher microvillar density than naive CD8+ T cells. Mapping the adhesion receptors CD2 and LFA-1 on the T cell surface reveals that CD2 exhibits a stronger preference for localizing to microvilli tips compared to TCR and LFA-1. Within microvilli, CD2 displays a strong spatial association with TCR clusters, contrasting with a markedly weaker TCR-LFA-1 association. At the IS, TCR and CD2 co-occupy close membrane contacts, while LFA-1 is excluded to more distal regions. Upon termination of activation, TCR clusters selectively disengage from microvilli, whereas CD2 and LFA-1 persist. These results suggest a coordinated, activation-dependent organization of adhesion receptors that drives microvillar adhesion during scanning and stabilizes IS membrane contacts. Together, NanoMAP establishes a powerful framework for dissecting nanoscale membrane architecture and spatial signaling in T cells.

immunology↗

bioRxiv2025

Identifying the therapeutic potential of Niclosamide in overcoming IFN-gamma dependent cancer immune evasion in the Tumor Microenvironment

BackgroundTumor cells frequently develop immune resistance through interferon-{gamma} (IFN-{gamma})-induced PD-L1 expression, acquisition of cancer stem cell (CSC)-like features, and adaptation to hypoxia within the tumor microenvironment (TME). Although IFN-{gamma} activates both STAT1 and STAT3, how these pathways interact to regulate immune evasion under hypoxia remains unclear. MethodsUsing the MC38 murine colorectal cancer model and T cell-tumor spheroid co-culture assays, we examined how IFN-{gamma} signaling through STAT1 and STAT3 influences PD-L1 expression, CSC plasticity, and cytotoxic T cell function under normoxic and hypoxic conditions. Pharmacologic inhibitors and siRNA knockdown were used to dissect pathway function, and Niclosamide, an FDA-approved anthelmintic, was evaluated as a dual STAT1/STAT3 inhibitor. ResultsWe found that IFN-{gamma} primarily induced PD-L1 through STAT1 activation, while CSC plasticity was associated with STAT3 signaling. STAT1 and STAT3 displayed reciprocal regulation--blocking one enhanced activation of the other. Niclosamide effectively inhibited phosphorylation of both STAT1 and STAT3, which led to suppressed PD-L1 upregulation and reduced CSC enrichment. In addition, it also partially inhibited hypoxia-induced HIF-1 expression. In co-culture assays, Niclosamide improved T cell infiltration and reduced exhaustion under hypoxic conditions, resulting in improved T cell killing. ConclusionsOur findings identified Niclosamide as a potent dual STAT1/3 inhibitor capable of reversing IFN-{gamma} and hypoxia-driven immune evasion. Repurposing Niclosamide may represent a promising strategy to enhance the efficacy of immune checkpoint blockade in solid tumors. key messagesInterferon-{gamma} (IFN-{gamma}) enhances cytotoxic T cell function but also promotes tumor immune evasion by upregulating PD-L1 and inducing cancer stem cell- like properties. Our study identifies a reciprocal regulatory mechanism between STAT1 and STAT3 in IFN-{gamma}-treated tumor cells that shapes immune evasion outcomes. We demonstrate that Niclosamide, an FDA-approved anthelmintic, acts as a dual STAT1/STAT3 inhibitor, effectively suppressing PD-L1 induction, limiting cancer stemness, and reducing HIF-1 expression under hypoxia. Niclosamide also restores T cell infiltration and decreases exhaustion in a 3D tumor spheroid model. By repurposing Niclosamide, this work provides a feasible approach to enhance the efficacy of immune checkpoint blockade and guide future translational and clinical studies in immunotherapies against solid tumors.

immunology↗

bioRxiv2025

Acute kidney injury disrupts cardiac remodeling via SerpinA3N

Cardiorenal syndrome type 3 (CRS-3) also known as acute reno-cardiac syndrome, refers to a condition where acute kidney injury (AKI) leads to acute cardiac dysfunction or injury. The underlying mechanisms have not been elucidated. In the current study, we examined the potential mechanisms for the development of myocardial pro-fibrotic factors and cardiac remodeling in C57BL/6J mice after AKI. The mice were subjected to bilateral pedicle clamping for 30 min followed by 24 h of reperfusion. Heart tissue was collected from control (Ctrl) and AKI mice and subjected to proteomic analysis by liquid chromatography-mass spectrometry (LC-MS) and differential mRNA expression analysis by RNA-seq. Cardiac tissue was collected to assess serine protease activity, RNA, protein and cytokine expression. The results indicate that AKI significantly upregulated SerpinA3N in the heart which was negatively correlated with serine protease (Granzyme B) activity. Further, AKI induced the development of several pro-fibrotic factors, induced mitochondrial dysfunction and increased inflammation. Finally, using H9C2 cells we demonstrated that inhibiting SerpinA3N with XAV939 increased Granzyme B activity. Thus, this study suggests that AKI leads to the development of pro-fibrotic factors in the heart and disrupts cardiac remodeling by SerpinA3N mediated inhibition of serine protease Granzyme B activity. Decreased SerpinA3N expression in the heart followed by AKI could lead to a more balanced extracellular matrix (ECM) composition in the heart with an alleviated pro-fibrotic response.

molecular biology↗

bioRxiv2025

Separable roles for Microprocessor and its cofactors ERH and SAFB1/2 during microRNA cluster assistance

While most conserved microRNA (miRNA) transcripts harbor a suite of features that mediate their efficient biogenesis into small RNAs, some loci bear suboptimal attributes that enable additional layers of processing regulation. A notable example is cluster assistance, whereby a miRNA hairpin with suboptimal nuclear biogenesis can be enhanced by an optimal neighbor. This process involves local transfer of the Microprocessor complex, composed of the RNase III enzyme Drosha and its partner DGCR8, in concert with cofactors such as ERH and SAFB1/2. However, the mechanism(s) that underlie miRNA cluster assistance remain largely unclear. Here, we gain insights into this process by integrating mutant cells of Microprocessor and its cofactors with analysis of miRNA structure-function variants, biochemical tests and genomewide profiling. We define features of suboptimal miRNAs that render them dependent on cluster assistance, and distinguish amongst a network of proposed interactions amongst Microprocessor and its cofactors, to reveal a subset that are critical for cluster assistance. Most importantly, we use epistatic tests to separate and order the functional requirements for ERH and SAFB1/2 into a pathway. Our data indicate that ERH may engage in the process of Microprocessor transfer between hairpins, while SAFB factors (especially SAFB2) mediate recognition and stable binding of a suboptimal miRNA hairpin after Microprocessor transfer. Finally, we show how cluster assistance integrates into a feedback regulatory loop on Microprocessor, via Drosha-mediated cleavage of a suboptimal miRNA hairpin in the DGCR8 transcript. Altogether, our findings reveal complex regulatory transactions during biogenesis of clustered miRNAs.

molecular biology↗

bioRxiv2020

The Exon Junction Complex and intron removal prevents resplicing of mRNA

Accurate splice site selection is critical for fruitful gene expression. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic splice sites (SS), even though majority of these bear weak splicing motifs and appear incompetent. Mechanistically, the EJC directly conceals splicing elements through position-specific recruitment, preventing SS definition. We note that intron removal using strong, canonical SS yields AG|GU signatures at exon-exon junctions. Unexpectedly, we discover that scores of these minimal exon junction sequences are in fact EJC-suppressed 5 and 3 recursive SS, and that loss of EJC regulation from such transcripts triggers faulty mRNA resplicing. An important corollary is that intronless cDNA expression constructs from aforementioned targets yield high levels of unanticipated, truncated transcripts generated by resplicing. Consequently, we conclude the EJC has ancestral roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

genomics↗

bioRxiv2019

BEN-solo factors partition active chromatin to ensure proper gene activation in Drosophila

The Drosophila genome encodes three BEN-solo proteins including Insensitive (Insv), Elba1 and Elba2 that possess activities in both transcriptional repression and chromatin insulation. These proteins all have a DNA binding BEN domain. A fourth protein Elba3 bridges Elba1 and Elba2 to form a heterotrimeric complex ELBA. Here we report comprehensive investigation on the in vivo functions of these proteins in Drosophila embryos. We generate ChIP-seq data of all these factors from all cognate and non-cognate mutants to assess common and distinct binding locations of Insv and ELBA, and genetic interdependencies. Notably, while most Elba1 and Elba2 binding requires the full ELBA complex, the adapter protein Elba3 can associate with chromatin and repress gene expression independently of Elba1 and Elba2. We also employ high-resolution ChIP-nexus mapping to show that Insv binds to DNA in a symmetric configuration while the ELBA complex binds asymmetrically in vivo. We observe that motifs of known insulator proteins are enriched in ELBA and Insv ChIP peaks and demonstrate that ELBA collaborates with other insulator factors to regulate developmental patterning in embryos. To differentiate the insulator function of ELBA and Insv from their repressor activity, we determined real-time transcription change in mutant embryos using precision nuclear run-on sequencing. ELBA factor mutants dampen expression differences between pairs of ELBA-bound neighboring genes. Finally, transgenic reporters confirm insulation activity of ELBA- and Insv-bound sites. Altogether, these findings define ELBA and Insv as general insulator proteins in Drosophila and demonstrate the functional importance of insulators in partitioning transcription units.

developmental biology↗

bioRxiv2018

Crossbrowse: A versatile genome browser for visualizing comparative experimental data

The recent beyond-exponential growth in diverse collections of deep sequencing datasets creates enormous opportunities for discovery, concomitant with new challenges for displaying and interpreting these data. Notably, the availability of scores of whole genome sequences in multiple species clades enables comparative studies of functional elements. However, current genome browsers do not permit effective visualization of multigenome experimental data. Here, we present CrossBrowse, a standalone desktop application for displaying and browsing cross-species genomic datasets. We utilize data standards and graphic representation of popular browsers, and incorporate an intuitive graphical visualization of genome synteny that facilitates and drives human interrogation of comparative data. Our platform permits users with minimal informatics capacity to select arbitrary sets of genomes for display, upload and configure multiple datasets, and interact with vertebrate-sized genomic datasets in real-time. We illustrate the utility of CrossBrowse with interrogation of comparative invertebrate and mammalian datasets that provide insights into diverse aspects of transcriptional and post-transcriptional regulation. Of note, we show examplars of both preservation and divergence of functional elements that cannot be inferred from sequence alignments alone. Moreover, we demonstrate how inspection of primary data using CrossBrowse exposes an artifact in a typical strategy for assigning species-specific functional elements, and drives the implementation of an improved computational strategy. We anticipate that CrossBrowse will greatly foster user-based discovery within multispecies genomic datasets, and inform their bioinformatic interpretation.

bioinformatics↗

bioRxiv2017

Deep experimental profiling of microRNA diversity, deployment, and evolution across the Drosophila genus

Comparative genomic analyses of microRNAs (miRNAs) have yielded myriad insights into their biogenesis and regulatory activity. While miRNAs have been deeply annotated in a small cohort of model organisms, evolutionary assessments of miRNA flux are clouded by the functional uncertainty of orthologs in related species, and insufficient data regarding the extent of species-specific miRNAs. We address this by generating a comparative small RNA (sRNA) catalog of unprecedented breadth and depth across the Drosophila genus, extending our extant deep analyses of D. melanogaster with sRNA data from multiple tissues of 11 other fly species. Aggregate analysis of several billion sRNA reads permits curation of accurate and holistic compendia of miRNAs across this genus, providing abundant opportunities to identify species- and clade-specific variation in miRNA identity, abundance, and processing. Amongst well-conserved miRNAs, we observe unexpected cases of clade-specific variation in 5' end precision, occasional antisense loci, and some putatively non-canonical loci. We also employ strict criteria to identify a massive set (649) of novel, evolutionarily-restricted miRNAs. Amongst the bulk collection of species-restricted miRNAs, two notable subpopulations of rapidly-evolving miRNAs are splicing-derived mirtrons and testis-restricted, clustered (TRC) canonical miRNAs. We quantify rates of miRNA birth and death using our annotation and a phylogenetic model for estimating rates of miRNA turnover in the presence of annotation uncertainty. We show striking differences in birth and death rates across miRNA classes defined by biogenesis pathway, genomic clustering, and tissue restriction, and even identify variation heterogeneity amongst Drosophila clades. In particular, distinct molecular rationales underlie the distinct evolutionary behavior of different miRNA classes. We broaden observations made from D. melanogaster as Drosophilid-wide principles for opposing evolutionary viewpoints for miRNA maintenance. Mirtrons are associated with a high rate of 3' untemplated addition, a mechanism that impedes their biogenesis, whereas TRC miRNAs appear to evolve under positive selection. Altogether, these data reveal miRNA diversity amongst Drosophila species and permit future discoveries in understanding their emergence and evolution.

genomics↗