Biology subjects

Hu, R.

Publications and source records attributed to Hu, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

A cytochrome P450 G subfamily member, CYP4G35, is highly expressed in antennae and modulates olfactory response in Aedes aegypti mosquitoes.

The cytochrome P450 enzymes of the CYP4G subfamily are some of the most enigmatic insect P450s. The dipterans with sequenced genomes have two CYP4G paralogs. In Drosophila melanogaster, CYP4G1 is highly expressed in the oenocytes and catalyzes the last enzymatic step in the biosynthesis of cuticular hydrocarbons. In contrast, CYP4G15 is expressed in the brain glial cells, but its function is unknown. The Aedes aegypti genome encodes two CYP4Gs: CYP4G36 (ortholog of DmCYP4G1) and CYP4G35 (ortholog of DmCYP4G15). Here, we show that CYP4G35 is highly expressed in mosquito antennae, and the RNAi knockdown of CYP4G35 results in delayed host-seeking. Ae. aegypti CYP4G knockout lines confirmed delayed host-seeking behavior in CYP4G35 knockout females. Proteomics analysis of CYP4G35 KO females also corroborates the physiological findings and shows upregulation of proteins related to olfaction and other CYP4Gs to compensate for the lack of CYP4G35. Immunohistochemistry and in situ hybridization were used to localize CYP4G35 and demonstrated its expression in the sensilla lymph of the antennae and the tip of the proboscis. CYP4G35 and CYP4G36 fusion proteins with cytochrome P450 reductase demonstrated that, unlike CYP4G36, CYP4G35 lacks an oxidative decarbonylase function. Together, our data support a novel function of CYP4G35 in modulating olfactory response.

molecular biology↗

bioRxiv2025

Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity

Cross-kingdom RNAs are emerging as critical mediators of interspecies interactions, yet the functions of long RNAs such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms remain largely unexplored. Here, we show that the brown planthopper (Nilaparvata lugens, BPH), a major rice pest, translocates mRNAs and lncRNAs into rice plants, where they migrate systemically from feeding sites to distal tissues. Compared with BPH mRNAs, BPH Salivary gland Cross-kingdom LncRNA (BSCLs) exhibit markedly higher stability in rice. Among them, BSCL1 functions as a virulence factor that promotes BPH feeding and reproduction by suppressing host defense. Mechanistically, BSCL1 associates with the HIRA histone chaperone complex and displaces histone H3.3 from the promoters of transcription factors, including bHLH genes central to jasmonic acid signaling, thereby repressing transcriptional immunity. Our results identify BSCLs as systemic, RNA-based effectors that reprogram host defense at the epigenetic level, revealing a previously unrecognized mode of insect-mediated manipulation of plant immunity and highlighting lncRNAs as cross-kingdom regulators. Significance StatementPlant-herbivore interactions are traditionally viewed as battles over nutrients and defense signaling, mediated largely by proteins and small RNAs. Here, we demonstrate that long non-coding RNAs from brown planthopper saliva are translocated into rice, migrate systemically, and function as epigenetic effectors that suppress key transcription factors in jasmonic acid-mediated defense. Unlike insect mRNAs, these lncRNAs persist in the plant and reprogram immunity by interfering with histone deposition. This discovery uncovers a new class of mobile, RNA-based virulence factors, expands our understanding of cross-kingdom regulation, and suggests innovative strategies for pest control targeting RNA effectors.

plant biology↗

bioRxiv2025

Systematic analysis of naturally occurring missense mutations in human manganese transporters: predictions and structural insights

Manganese (Mn) homeostasis in humans is tightly regulated by the transporters ZIP8, ZIP14, and ZnT10, and pathogenic mutations in these proteins cause systemic Mn dysregulation, leading to severe disorders in multiple systems. Here, we performed a systematic survey of naturally occurring missense variants in these transporters. Pathogenicity was assessed with two widely used computational tools, CADD and AlphaMissense (AM), and results were integrated with AlphaFold-predicted structural models. Although the prediction methods showed general agreement, substantial discrepancies were observed, with CADD tending to overpredict deleteriousness and AM failing to identify a portion of confirmed pathogenic variants. Structural mapping revealed that predictions were more accurate for amino acid substitutions in structured and buried residues than for solvent-exposed loops and flexible regions. Mechanistic insights further highlighted variants at functionally important regions as critical. Our study helps prioritize Mn transporter variants for future experimental validation and provides a framework for bridging computational predictions with structure-guided mechanistic studies.

biochemistry↗

bioRxiv2025

Mycobacterium tuberculosis, Mycobacterium kansasii and Rhodococcus equi induce macrophage necroptosis in the presence of a caspase inhibitor acting on a non-canonical target(s)

Macrophages are the predominant cell type infected by Mycobacterium tuberculosis (Mtb) in tuberculosis (TB). Death of Mtb-infected macrophages promotes tissue pathology and releases Mtb to infect other cells, suggesting that inhibiting the death of Mtb-infected macrophages could be an adjunctive treatment of TB. Prospects for such an intervention depend on identifying the molecular pathways leading to cell death. We previously reported that the death of Mtb-infected mouse macrophages in vitro depends on type I interferon (IFN) and that the ensuing upregulation of cis-aconitate decarboxylase (ACOD1; IRG1) contributes to cell death by exacerbating Mtb-induced lysosomal membrane permeabilization. Here we report that death of Mtb-infected primary mouse macrophages in vitro became necroptotic and die faster in the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD) acting on a target other than caspase-8. Macrophages infected with Mycobacterium kansasii and Rhodococcus equi likewise underwent z-VAD-dependent necroptosis. In C57BL/6 mice, which are relatively TB-resistant, we saw no impact of MLKL deficiency on bacterial burden or pulmonary pathology. In contrast, in Sp140-/- mice on the C57BL/6 background, which express high levels of type I IFN after Mtb infection and develop necrotic pulmonary lesions, MLKL-deficiency reduced bacterial burden and pathology after high-dose infection. This report illustrates that off-target action(s) of a caspase-8 inhibitor can switch the cell death pathway to necroptosis in macrophages infected with various Gram-positive pathogens. In turn, this opens the possibility that pathophysiologic circumstances may lead to inhibition of the same target(s) that z-VAD inhibited in our studies. That may be what allows MLKL to exacerbate tuberculosis in mice that are prone to formation of necrotic lesions. Author SummaryTuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), led to about 1.3 million deaths in 2023. The rapid emergence of drug resistance and slow pace of new drug development prompt attention to adjunctive host-directed therapies, an approach that relies on a thorough and detailed understanding of host-Mtb interactions. Mtb infection of macrophages can kill them, promoting tissue pathology and releasing of Mtb to infect other cells. Here we find that a pan-caspase inhibitor z-VAD hastened the death of Mtb-infected macrophages and converted the mode of cell death to RIPK1-RIPK3-MLKL-dependent necroptosis. However, the functional target(s) of z-VAD-FMK and the activation mechanism of RIPK3 differ from the canonical pathways. The pan-caspase inhibitor also promoted rapid, necroptotic death of macrophages infected with Mycobacterium kansasii and Rhodococcus equi. We further found that MLKL-deficiency in Sp140-/- mice on the C57BL/6 background resulted in less weight loss, lower Mtb burdens and mitigation of lung pathology after high-dose Mtb infection. Our findings suggest that necroptosis can contribute to TB pathogenesis in some circumstances. Further identification of the death pathway-switching mechanism could deepen our understanding of host-Mtb interactions and aid in the design of adjunctive host-directed therapies.

microbiology↗

bioRxiv2025

Fast Multi-objective RNA Optimization with Autoregressive Reinforcement Learning

Codon optimization is essential in mRNA vaccine development, while existing tools face limitations in the computational efficiency, sequence diversity and universality. To address these challenges, we develop RNAJog (RNA Joint Optimization with autoregressive Generative model), a framework integrating autoregressive generation with reinforcement learning to optimize codon sequences for minimum free energy (MFE), codon adaptation index (CAI) and GC content, even enabling sequence design without requiring annotated training data. Evaluations in both in silico and wet-lab experiments have confirmed RNAJogs effectiveness and efficiency, with two orders of magnitude faster than traditional algorithm (LinearDesign) for long RNA sequence and about a 10-fold increase in antibody titer compared to the wild-type mRNA for Influenza virus hemagglutinin (HA) mRNA vaccine design in mouse. RNAJog also supports biological constraints for sequence optimization. Using this feature, we minimized m6A modification motifs in Bmp2 coding sequence for enhancing the translational efficiency and RNA stability, which are validated in cell-based experiments.

bioinformatics↗

bioRxiv2025

Lithosyntrophy: Obligate syntrophy in a phosphite-oxidizing, methanogenic culture

The anaerobic conversion of organic matter to methane and carbon dioxide typically relies on obligate syntrophic interactions between bacteria and methanogenic archaea, where interspecies hydrogen (H2) transfer enables thermodynamically constrained reactions to proceed near equilibrium. Syntrophs couple the oxidation of fermentation products such as fatty acids and alcohols to the reduction of protons to form H2. These reactions can only proceed if low H2 concentrations are maintained by H2-consuming syntrophic partners. Here, we describe "lithosyntrophy," a novel mode of syntrophic interaction in which electrons that drive hydrogenotrophic methanogenesis originate from an inorganic compound rather than from the canonical organic substrates. Candidatus Phosphitivorax anaerolimi strain Phox-21 oxidizes phosphite (HPO32-, oxidation state +3) to phosphate coupled to hydrogenogenesis in an obligate energetic dependency on a hydrogenotrophic methanogen, Methanoculleus sp. Physiology experiments, thermodynamic calculations, genomic annotation, and metaproteomics analysis collectively revealed a mechanism for syntrophic phosphite oxidation in Phox-21, which requires phosphite, acetate, and CO2 as co-substrates. In this pathway, electrons derived from phosphite drive H2 production via an electron-confurcating hydrogenase. Unlike previously characterized acetogenic phosphite oxidizers that grow without exogenous acetate, Phox-21 requires acetate to regenerate AMP, a cofactor required by the phosphite dehydrogenase, PtdF. Lithosyntrophic phosphite oxidizers may play important roles both in transferring reducing equivalents as well as biologically available phosphorus to other members of their surrounding microbial communities. We infer that lithosyntrophic DPO emerged before acetoclastic methanogenesis and was a major sink for acetate in the Archaean when phosphite was more abundant. Significance statementDissimilatory phosphite-oxidizing microorganisms (DPOM) use phosphite as an energy source, producing phosphate. While the two previously isolated DPOM couple phosphite oxidation to carbon fixation via the Wood-Ljungdahl pathway, Candidatus Phosphitivorax anaerolimi strain Phox-21 instead performs lithosyntrophic metabolism, coupling DPO to proton reduction in obligate partnership with a hydrogenotrophic methanogen. This establishes a novel link between phosphorus and carbon redox cycles: like organosyntrophy, DPO can fuel methanogenesis. Lithosyntrophy expands our understanding of syntrophic interactions and suggests similar processes may occur with other inorganic substrates in anoxic ecosystems. We propose lithosyntrophic DPO predates acetoclastic methanogenesis, serving as an early acetate sink during the Archaean. Following Earths oxygenation, phosphite depletion likely reduced competition for acetate, enabling the later evolution of acetoclastic methanogenesis.

microbiology↗

bioRxiv2025

Poldip2 deficiency attenuates disease severity in a mouse model of COVID-19

The lungs are the primary target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2), with the infection resulting in lung inflammation, pulmonary vascular leakage and diffuse alveolar damage. Polymerase delta-interacting protein-2 (Poldip2) mediates lung inflammation and vascular permeability after lipopolysaccharide-induced acute respiratory distress syndrome; however, its role in regulating lung permeability, vascular inflammation and tissue damage following SARS-CoV-2 infection is completely unknown. Here, we assessed the role of Poldip2 in inflammation, immune cell infiltration and lung tissue damage in response to SARS-CoV-2 infection. Our data shows that while deletion of Poldip2 does not affect the susceptibility to SARS- CoV-2 infection, mice heterozygous for Poldip2 exhibit reduced lung tissue damage, reduced cytokine and chemokine induction and decreased infiltration of myeloperoxidase (MPO)-positive neutrophils into inflamed lung tissue. These data reveal that Poldip2 depletion mitigates inflammation and immune cell infiltration following SARS-CoV-2 infection, highlighting the therapeutic potential of Poldip2 inhibition to attenuate severe lung injury.

pathology↗

bioRxiv2025

Induction of Human Pruriceptors from Pluripotent Stem Cells via Transcription Factors

Pruriception is crucial for defense against external stimuli but can lead to chronic pruritus, a debilitating condition affecting millions worldwide. Our understanding of the cellular and molecular mechanisms behind the sensation of itch has been hindered by the lack of functional human models. Here, we address this limitation by developing a protocol to generate induced pruriceptors (iPruriceptors) from human pluripotent stem cells (hPSCs). We compared two differentiation approaches: a direct method via forced expression of transcription factors (TFs) in hPSCs, and a 2-step process through expression of TFs in hPSC-derived neural crest cells (NCCs). The 2-step protocol proved superior in inducing a transcriptional program that closely resembles that of human pruriceptors. Our optimized protocol employs forced expression of NGN1 and ISL1 to drive differentiation from NCCs into pruriceptors, enhancing the expression of known pruritogen receptors such as IL31RA, which pairs with OSMR, and HRH1. The induction of this transcriptional program leads to functional maturation of iPruriceptors. Accordingly, iPruriceptors exhibit robust responses to itch stimuli and in vivo-like itch pharmacology such as treatment with ABT-317, a JAK1 inhibitor tool compound, similar to those targeting intensive pruritus in atopic dermatitis (AD). Importantly, iPruriceptors can be generated without viral vectors or safe-harbor gene editing, using a PiggyBac-based transfection method that simplifies scalability. Our protocol offers a robust platform for investigating itch biology, modeling chronic pruritus, and enabling high-throughput screening for therapeutic target discovery. HighlightsO_LINGN1 and ISL1 forced expression in NCCs induces rapid differentiation to iPruriceptors C_LIO_LIiPruriceptors share transcriptional profile of primary human pruriceptors C_LIO_LIiPruriceptors have electrophysiological responses to known pruritogens C_LIO_LIiPruriceptors have JAK1-dependent IL-31/-13 responses blocked by ABT-317 C_LI

neuroscience↗

bioRxiv2025

StringTie3 Improves Total RNA-seq Assembly by Resolving Nascent and Mature Transcripts

Accurate assembly of rRNA-depleted (total) RNA-seq remains challenging because existing methods often conflate incomplete, nascent RNA with fully processed mature isoforms, leading to misassemblies and quantification errors that skew downstream analyses. Here, we present StringTie3, a major update to the widely used StringTie assembler, specifically designed for total RNA-seq. This new version introduces two key innovations: (1) a nascent mode that models co-transcriptional splicing to separate nascent from mature transcripts, and (2) a refined long-read module that distinguishes genuine polyadenylation sites from poly(A)-priming artifacts. Across short-, long-, and hybrid-read datasets, StringTie3 substantially reduces assembly errors and outperforms existing tools, boosting precision by up to 20% in short-read total RNA-seq and improving sensitivity and precision by as much as 37% and 75%, respectively, in long-read assemblies. In Argonaute knockout experiments, nascent-mode analysis shows that single knockouts predominantly alter nascent transcripts while leaving mature RNA largely unchanged, whereas double or triple knockouts disrupt both fractions. Applying this approach to breast cancer samples shows that, although nascent and mature RNA levels often correlate, certain extracellular matrix and tumor suppressor genes deviate from this pattern, suggesting post-transcriptional regulation. By accurately reconstructing transcriptomes and distinguishing nascent from mature RNA, StringTie3 reveals hidden layers of RNA regulation and provides a powerful framework for investigating transcriptional and post-transcriptional processes in total RNA-seq data.

genomics↗

bioRxiv2025

Deregulated Myt3 translation predisposes islet β-cells to dysfunction under obesity-induced metabolic stress

In response to obesity-related metabolic stress, islet {beta}-cells adapt (or compensate) by increasing their secretory function and mass. Yet, for unknown reasons, this compensation is reversed in some individuals at some point to induce {beta}-cell failure and overt type 2 diabetes (T2D). We have previously shown that transcription factor Myt3 (St18) and its paralogs, Myt1 and Myt2, prevent {beta}-cell failure. Myt3 was induced at post-transcriptional levels by obesity-related stress in both mouse and human {beta} cells and its downregulation accompanied {beta}-cell dysfunction during T2D development. Single-nucleotide polymorphisms in MYT3 were associated with an increased risk of developing human diabetes. We now demonstrate that Myt3 translation is regulated by an upstream open-reading frame that overlaps with the main Myt3 open-reading frame in mice. Disrupting this overlap enhances Myt3 translation in mouse {beta} cells without metabolic stress but decreases it under high-fat-diet challenges. Consequently, this deregulation results in {beta}-cell dysfunction and glucose intolerance in mice, accompanied by compromised expression of several {beta}-cell function genes. These findings suggest that stress-induced Myt3 translation is part of the compensation mechanism that prevents {beta}-cell failure.

genetics↗

bioRxiv2025

Organic carbon oxidation state shapes fermentative methanogenic microbiomes and controls greenhouse gas fluxes

Organic compounds with a negative nominal oxidation state of carbon (NOSC) are thermodynamically recalcitrant in anaerobic ecosystems, but few studies have measured the influence of NOSC on carbon degradation rates, gaseous product yields, or microbiome composition. We amended anaerobic rice paddy sediment microcosms with monomeric organic carbon compounds varying in NOSC. Consistent with thermodynamic and stoichiometric predictions, negative NOSC compounds are catabolized more slowly but produce more methane per mole of carbon. Negative NOSC microbiomes have higher alpha diversity, more syntrophs and methanogens, and fewer fermentative bacteria. Strikingly, fermentative bacterial taxa display genomically encoded NOSC catabolic preferences both in the lab and field. Negative NOSC- preferring fermenters have longer predicted doubling times, consistent with the thermodynamic recalcitrance of their preferred substrates. We propose that microbial NOSC preference can be leveraged for predicting and engineering greenhouse gas fluxes and understanding bacterial population dynamics and trait evolution across redox gradients.

microbiology↗

bioRxiv2025

Integrated single-cell and spatial analysis identifies context-dependent myeloid-T cell interactions in head and neck cancer immune checkpoint blockade response

BackgroundApproximately 15-20% of head and neck cancer squamous cell carcinoma (HNSCC) patients respond favorably to immune checkpoint blockade (ICB). Previous single-cell RNA-Seq (scRNA-Seq) studies identified immune features, including macrophage subset ratios and T-cell subtypes, in HNSCC ICB response. However, the spatial features of HNSCC-infiltrated immune cells in response to ICB treatment need to be better characterized. MethodsHere, we perform a systematic evaluation of cell interactions between immune cell types within the tumor microenvironment using spatial omics data using complementary techniques from both 10X Visium spot-based spatial transcriptomics and Nanostring CosMx single-cell spatial omics with RNA gene panel including 435 ligands and receptors. In this study, we used integrated bioinformatics analyses to identify cellular neighborhoods of co-localizing cell types in single-cell spatial transcriptomics and proteomics data. In addition, we used both publicly available scRNA-Seq and in-house spatial RNA-Seq data to identify spatially constrained Ligand-Receptor interactions in Responder patients. ResultsWith 522,399 single cells profiled with both RNA and protein from 26 patients, in addition to spot-resolved spatial RNA-Seq from 8 patients treated with ICB together with bioinformatics analysis of publicly available single-cell and bulk RNA-Seq, we have identified a spatial and cell-type specific context-dependency of myeloid and T cell interaction difference between Responders and Non-Responders. We defined further cellular neighborhood and the sources of chemokine CXCL9/10-CXCR3 interactions in Responders, emerging targets in ICB, as well as CXCL16-CXCR6, CCL4/5-CCR5, and other underappreciated and potential markers and targets for ICB response in HNSCC. In addition, we have contributed a rich data resource of cell-cell Ligand Receptor interactions for the immunotherapy and HNSCC research community. DiscussionOur work provides a comprehensive single-cell and spatial atlas of immune cell interactions that correlate with response to ICB in HNSCC. We showcase how integrating multiple technologies and bioinformatics approaches can provide new insights into potential immune-based biomarkers of ICB response. Our results suggested refining future studies using preclinical animal models in a more context-specific manner to elucidate potential underlying mechanisms that lead to improved ICB responses. What is already known on this topicMost cancer patients still do not experience clinical benefits from immune checkpoint blockade (ICB), necessitating the development of response biomarkers and new immunotherapeutic targets. What this study addsHere, we use integrated high-dimensional omics and bioinformatics approaches to identify immune cell-cell interaction markers associated with ICB response in patients with Head and neck squamous cell carcinoma. How this study might affect research, practice or policyWe identified spatial and cell-type specificity of Ligand-Receptor interactions between myeloid and T cells in ICB Responder patients that may help inform further mechanistic studies and biomarker development.

cancer biology↗

bioRxiv2025

Coordinated horizontal transfer of multiple genes assembles a carotenoid biosynthesis pathway in aphids

Horizontal gene transfer (HGT) plays a crucial role in genome evolution, especially when it enables the acquisition and assembly of multi-step biosynthetic pathways. Here, we investigate the evolutionary origins of carotenoid biosynthesis genes in aphids to determine whether multiple functionally related genes were acquired through HGT. We analyzed carotenoid biosynthesis genes in 23 aphid genomes based on homologs in plants, fungi, and bacteria. Phylogenetic analyses revealed that Geranylgeranyl pyrophosphate synthase (GPS), Phytoene synthase (PS), and Carotenoid desaturase (CD) were acquired via HGT from fungi by ancestral insect species, while Carotenoid cleavage oxygenase (CCO) appears to be a native insect gene. Most insect genomes contain two GPS copies, likely resulting from independent HGT events, whereas aphid genomes exhibit extensive duplication of PS and CD, a pattern uncommon in other insects. Expression analyses across aphid species with distinct pigmentation showed that these genes are broadly transcribed with substantial variability in expression levels. In Myzus persicae, comparative expression analysis between reddish and greenish clones, as well as a green-reddish clone with green and red color polymorphism, revealed that PS-4390 is a novel contributor to red pigmentation in M. persicae, in addition to CD-4400, a homolog of the tor gene in Acyrthosiphon pisum. These findings provide strong evidence that HGT can introduce multiple functionally related genes into recipient genomes, allowing them to be co-opted into a functional biosynthetic pathway.

evolutionary biology↗

bioRxiv2025

Probing the substrate binding-induced conformational change of a ZIP metal transporter using a sandwich ELISA

Zrt-/Irt-like proteins (ZIPs), a family of divalent metal transporters, are crucial for maintaining the homeostasis of zinc, an essential trace element involved in numerous biological processes. While extensive research on the prototypical ZIP from Bordetella bronchiseptica (BbZIP) have suggested an elevator transport mechanism, the dynamic conformational changes during the transport cycle have not been thoroughly studied. In this work, we developed a sandwich ELISA using a custom anti-BbZIP monoclonal antibody to investigate the conformational change induced by the metal binding to the transport site. This was achieved by determining the accessibility of a cysteine residue introduced at a position exposed to the solvent only when the transporter adopts an outward-facing conformation. This assay allowed us to report the dissociation constants of BbZIP for Zn2+ and Cd2+ at low and sub-micromolar levels, respectively. Notably, the installation of a positive charge at the M2 site drastically reduced metal binding at the M1 site, consistent with an auxiliary role for the M2 site in metal transport. We also demonstrated that this assay can be used to rapidly screen variants for subsequent structural study. We anticipate that other transporters where substrate binding induces large conformational changes can also be studied using this method.

biochemistry↗

bioRxiv2025

Bivalent ligands as a new universal chemotype of BCL6 degrader by inducing aggregation

BCL6 naturally exists as a homodimer to exert its transcriptional repressive function. Previous studies shown that molecular glue BI3802 can induce BCL6 dimer polymerization and thus degradation by ubiquitin-proteasome system. In this study we proposed a new strategy to degrade BCL6 by its homo-bivalent ligands, which can be rationally designed by assemble two BCL6 BTB ligands via linker. These homo-bivalent degraders (HBiDs) can induce polymerization of BCL6 dimer through chain assembly and ultimately lead to its ubiquitination and degradation. Since HBiD does not require a fragment to interact with components of protein degradation system, it could exhibit better target selectivity than traditional protein degraders while avoid drug resistance caused by mutations in protein degradation components. Here we will provide the proof-of-concept evidences for HBiD as BCL6 degrader. Most HBiDs synthesized in this study could degrade BCL6 regardless of the chemotype of BCL6 ligands, with extraordinary target selectivity. The degradation of BCL6 depends on the aggregation of BCL6 dimer induced by HBiD and the recruitment of SIAH1, the natural E3 ligase of BCL6. As protein dimerization is a common phenomenon in cells, HBiD may provide us with a powerful strategy for designing protein degraders as drug candidates or molecular probes.

biochemistry↗

bioRxiv2025

Myelin transcription factors 1 and 3 have overlapping but distinct roles in insulin secretion and survival of human β cells

Aims/hypothesisGenetic and environmental factors work together to cause islet beta-cell failure, leading to type 2 diabetes (T2D). How these factors are integrated to regulate beta cells remains largely unclear. Based on our previous findings that the family of Myelin transcription factors (MYT1, MYT1L, and ST18) prevents mouse beta-cell failure by repressing the overactivation of stress response, their regulation by obesity-related nutrition signals in human beta cells, and their association with T2D, we postulate that these factors prevent human beta-cell failure under normal physiology and obesity-related stress. MethodsMYT1 or ST18 were knocked down in primary human beta cells using shRNA. Beta-cell survival, secretory function, and gene expression were examined after islet cells were cultured in vitro or xenotransplanted into mice under normal or obesity-related stress. ResultsIn culture, MYT1-knockdown (KD) caused beta-cell death, while ST18-KD compromised glucose-stimulated insulin secretion. Under obesity-induced stress as xenotransplants, ST18-KD also caused beta-cell death. Accordingly, MYT1-KD deregulated several genes and genesets in cell death and cellular stress response, while ST18-KD deregulated those regulating stress response, mitochondria, and ion channels. Corresponding to these gene expression changes, ST18-KD reduces glucose-stimulated Ca2+ influx in beta cells. In addition, the MYT1- and ST18-regulated genes are enriched for T2D-associated loci, with an enrichment of 2.05-fold relative to random distribution. Conclusions/interpretationThe MYT TFs complement each other to integrate genetic and environmental factors to prevent beta-cell failure and T2D, with their major effects exerted on beta-cell viability and/or Ca2+ influx. Graphical AbstractNutrient-responsive transcription factors MYT1 and ST18 regulate human beta-cell survival and secretory functions. Under low metabolic stress, MYT1 regulates cell survival while ST18 regulates insulin secretion. Under high metabolic stress, ST18 also regulates beta-cell survival. MYT1, at least partly, regulates cell survival through stress-related apoptotic processes, while ST18 regulates insulin secretion via Ca2+ influx. Research in context summaryO_ST_ABSBackgroundC_ST_ABSO_LIThe myelin transcription factors (MYT TFs, including MYT1, MYT1l, and ST18) prevent mouse beta-cell failure by depressing the overactivation of stress-response genes. C_LIO_LISNPs in all three MYT loci are associated with human type 2 diabetes. C_LIO_LIThe expression and nuclear localization of MYT1 and ST18 were increased in primary human beta cells under acute metabolic stress but downregulated in type 2 diabetes. C_LIO_LIThe co-knockdown of MYT1, MYT1L, and ST18 in a human beta-cell line resulted in apoptosis. C_LI Key questionO_LIHow does MYT1 or ST18 regulate human beta-cell function and survival? C_LI New findingsO_LIMYT1 prevents human beta-cell death under normal and metabolic stress conditions, corresponding to deregulation of a few genes involved in cell death under cellular stress. C_LIO_LIST18 promotes insulin secretion under normal physiological conditions by regulating Ca2+ influx and prevents beta-cell death under metabolic stress. C_LIO_LIThe MYT1- and ST18-regulated genes are enriched for type 2 diabetes-risk loci. C_LI Clinical impactO_LIRegulators of the MYT-TF activities could be explored to delay/prevent beta-cell failure and the development of type 2 diabetes. C_LI

cell biology↗

bioRxiv2024

Characterizing postoperative T and B cell dysfunction in cancer surgery patients, using COVID-19 as a model antigen

For most cancers, surgery is an effective intervention method for cure but, despite the benefits, many patients recur with metastatic disease. Surgery has been shown to impair immune function by causing suppression of immune cells. In this study, we used a COVID-19 immunological toolkit to answer questions regarding the effects of surgery on antigen specific CD8+ T cells and B cells by exploiting the responses to the spike protein in vaccinated cancer patients. We demonstrate that surgical stress results in a reduction in the number of CD8+ T cell that produce cytokines and B cells that secrete antibodies in response to antigen. This study will improve our understanding of surgery-induced T cell and B cell dysfunction

immunology↗

bioRxiv2024

Expanding GABAergic Neuronal Diversity in iPSC-Derived Disease Models

GABAergic interneurons comprise diverse molecular and functional subtypes that contribute to neural circuit assembly and are implicated in a range of neurodevelopmental and neuropsychiatric disorders. Traditional approaches for differentiating human pluripotent stem cells (PSCs) into neurons often face challenges such as incomplete neural differentiation, prolonged culture periods, and variability across PSC lines, which can constrain their application in disease modeling. To address these limitations, we developed a strategy that combines inducible expression of the transcription factors (TFs) ASCL1 and DLX2 with dual-SMAD and WNT inhibition to efficiently drive differentiation of human PSCs into diverse, region-specific GABAergic neuronal types. Using single-cell sequencing, we characterized the cellular heterogeneity of GABAergic induced neurons (iNs) generated with the patterning factors (patterned iNs) and those derived solely with TFs (PSC-derived iNs), revealing distinct interneuron subtype compositions and the regulatory programs that govern their fate specification. Patterned iNs exhibited gene expression features corresponding to multiple brain regions, particularly the ganglionic eminence (GE) and neocortex, whereas GABAergic PSC-derived iNs predominantly resembled hypothalamic and thalamic neurons. Both iN types were enriched for genes relevant to neurodevelopmental and psychiatric disorders, with patterned iNs more specifically linked to neural lineage genes, highlighting their utility for disease modeling. We further applied this protocol to investigate the impact of a recurrent ADNP syndrome-associated mutation (p.Tyr719*) on GABAergic neuron differentiation, revealing that this mutation disrupts GABAergic fate specification and synaptic transmission. Overall, this study expands the toolkit for disease modeling by demonstrating the complementary advantages of GABAergic PSC-derived iNs and patterned iNs in representing distinct GABAergic neuron subtypes, brain regions, and disease contexts. Together, these approaches provide a flexible platform for investigating molecular and cellular mechanisms relevant to neurodevelopmental and neuropsychiatric disorders.

developmental biology↗