Biology subjects

Hu, X.

Publications and source records attributed to Hu, X..

At least 163 records · Page 9Linked to original sources

bioRxiv2024

A comprehensive collection of pain and addiction targets and compounds library for high throughput screening and AI-driven drug discovery

As part of the NIH Helping to End Addiction Long-term (HEAL) Initiative, the National Center for Advancing Translational Sciences (NCATS) is dedicated to the development of new pharmacological tools and investigational drugs for managing and treating pain, as well as the prevention and treatment of opioid misuse and addiction. In line with these objectives, we created a comprehensive, annotated small molecule library including drugs, probes, and tool compounds that act on published pain and addiction-relevant targets. Nearly 3,000 small molecules associated with approximately 200 known and hypothesized HEAL targets have been assembled, curated, and annotated in one collection. Physical samples of the library compounds have been acquired and plated in 1536-well format, enabling rapid and efficient high throughput screen (HTS) against a wide range of assays. The creation of the HEAL Targets and Compounds Library, coupled with an integrated computational platform for AI-driven machine learning (ML), structural modeling, and virtual screening (VS), provides a valuable source for strategic drug repurposing, innovative profiling, and hypothesis testing of novel targets related to pain and opioid use disorder (OUD). The library is available to investigators for screening pain and OUD-relevant phenotypes.

bioinformatics↗

bioRxiv2024

PSMA-Guided PROTAC Degraders for Tumor-Specific Protein Degradation in Prostate Cancer

PROTACs, degrading target protein to treat diseases, represent a highly promising drug design strategy. However, the degradation of target proteins by PROTACs in non-disease tissues may lead to systemic toxicity. Herein, capitalizing on the characteristic overexpression of PSMA in prostate cancer tumor tissues, we devised a PSMA-guided PROTACs specific targeting to prostate cancer. By conjugating AR degraders and BET degraders separately with PSMA ligands via cleavable linkers, two classes of PSMA-guided PROTACs were obtained. In vitro experiments demonstrated that PSMA-guided PROTAC molecules selectively degraded target proteins in PSMA-overexpressing prostate cancer cells, without affecting target proteins in non-PSMA-overexpressing cells. In vivo studies revealed that compared to conventional PROTACs, PSMA-guided PROTACs enhanced drug exposure in prostate cancer tumor tissues, prolonged half-life, and consequently achieved stronger and more sustained therapeutic effects. The PSMA-guided PROTAC strategy provides a novel avenue for disease tissue-specific PROTAC research, holding significant implications for targeted therapy in prostate cancer.

cancer biology↗

bioRxiv2024

Late-Stage Skeletal Muscle Transcriptome in Duchenne muscular dystrophy shows a BMP4-Induced Molecular Signature

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease due to loss-of-function mutations in the DYSTROPHIN gene. DMD-related skeletal muscle wasting is typified by an aberrant immune response involving upregulation of TGF{beta} family of cytokines. We previously demonstrated that bone morphogenetic protein 4 (BMP4) is increased in DMD and BMP4 stimulation induces a 20-fold upregulation of Smad8 transcription. However, the role of BMP4 in severely affected DMD skeletal muscle is unknown. We hypothesized that transcriptomic signatures in severely affected human DMD skeletal muscle are driven by BMP4 signaling. Transcriptomes from skeletal muscle biopsies of late-stage DMD vs. non-DMD controls and C2C12 muscle cells with or without BMP4 stimulation were generated by RNA-Seq and analyzed for single transcript differential expression as well as by Ingenuity Pathway Analysis and weighted gene co-expression network analyses. A total of 2,328 and 5,291 transcripts in the human muscle and C2C12 muscle cells, respectively, were differentially expressed. We identified an overlapping molecular signature of 1,027 genes dysregulated in DMD muscle that were induced in BMP4-stimulated C2C12 muscle cells. Highly upregulated DMD transcripts that overlapped with BMP4-stimulated C2C12 muscle cells included ADAMTS3, HCAR2, SERPING1, SMAD8, and UNC13C. The DMD transcriptome was characterized by dysregulation of pathways involving immune function, extracellular matrix remodeling, and metabolic/mitochondrial function. In summary, we define a late-stage DMD skeletal muscle transcriptome that substantially overlaps with the BMP4-induced molecular signature in C2C12 muscle cells. This supports BMP4 as a disease-driving regulator of transcriptomic changes in late-stage DMD skeletal muscle and expands our understanding of the evolution of dystrophic signaling pathways and their associated gene networks that could be explored for therapeutic development.

bioinformatics↗

bioRxiv2024

PLM_Sol: predicting protein solubility by benchmarking multiple protein language models with the updated Escherichia coli protein solubility dataset

Protein solubility plays a crucial role in various biotechnological, industrial and biomedical applications. With the reduction in sequencing and gene synthesis costs, the adoption of high-throughput experimental screening coupled with tailored bioinformatic prediction has witnessed a rapidly growing trend for the development of novel functional enzymes of interest (EOI). High protein solubility rates are essential in this process and accurate prediction of solubility is a challenging task. As deep learning technology continues to evolve, attention-based protein language models (PLMs) can extract intrinsic information from protein sequences to a greater extent. Leveraging these models along with the increasing availability of protein solubility data inferred from structural database like the Protein Data Bank (PDB), holds great potential to enhance the prediction of protein solubility. In this study, we curated an Updated Escherichia coli (E.coli) protein Solubility DataSet (UESolDS) and employed a combination of multiple PLMs and classification layers to predict protein solubility. The resulting best-performing model, named Protein Language Model-based protein Solubility prediction model (PLM_Sol), demonstrated significant improvements over previous reported models, achieving a notable 5.7% increase in accuracy, 9% increase in F1_score, and 10.4% increase in MCC score on the independent test set. Moreover, additional evaluation utilizing our in-house synthesized protein resource as test data, encompassing diverse types of enzymes, also showcased the superior performance of PLM_Sol. Overall, PLM_Sol exhibited consistent and promising performance across both independent test set and experimental set, thereby making it well-suited for facilitating large-scale EOI studies. PLM_Sol is available as a standalone program and as an easy-to-use model at https://zenodo.org/doi/10.5281/zenodo.10675340.

bioinformatics↗

bioRxiv2024

Circular DNA vcDNA-S7 derived from segmented-dsRNA virus BmCPV attenuated viral infection through RNase H1

Viral circDNA derived from insect positive-sense single-stranded RNA viruses plays a crucial role in regulating viral infections depending on the RNAi pathway. Our previous research has revealed that Bombyx mori cypovirus (BmCPV), a segmented-dsRNA virus, can generate viral circDNA vcDNA-S7, which can be transcribed to form RNA and further processed into small RNAs to defend against the virus through RNAi. However, whether vcDNA-S7 employs other mechanisms to resist the viruses. Presently, vcDNA-S7 has been found to resist BmCPV infection via a novel mechanism that depends on ribonuclease RNase H1 activity. It was found that the expression level of RNase H1 increased upon BmCPV infection (RNA virus) but not upon B. mori nucleopolyhedrovirus (BmNPV) (DNA virus) infection. RNase H1 has been shown to negatively regulate BmCPV infections. In addition, vcDNA-S7 was found to form a heteroduplex with the viral RNA and subsequently recruit RNase H1 to degrade the viral RNA within the heteroduplex, ultimately leading to the suppression of viral replication. Furthermore, the resistance of vcDNA-S7 to viruses was significantly reduced when cellular RNase H1 gene was silenced. These findings not only reveal a new antiviral mechanism involving circular DNA formed by reovirus but also offer intriguing insights into the antiviral function of RNase H1 in cells.

immunology↗

bioRxiv2024

Highly Pathogenic Avian Influenza A (H5N1) clade 2.3.4.4b Virus detected in dairy cattle

The global emergence of highly pathogenic avian influenza (HPAI) A (H5N1) clade 2.3.4.4b viruses poses a significant global public health threat. Until March 2024, no outbreaks of this virus clade had occurred in domestic cattle. We genetically characterize HPAI viruses from dairy cattle showing an abrupt drop in milk production. They share nearly identical genome sequences, forming a new genotype B3.13 within the 2.3.4.4b clade. B3.13 viruses underwent two reassortment events since 2023 and exhibit critical mutations in HA, M1, and NS genes but lack critical mutations in PB2 and PB1 genes, which enhance virulence or adaptation to mammals. The PB2 E627K mutation in a human case underscores the potential for rapid evolution post-infection, highlighting the need for continued surveillance to monitor public health threats.

microbiology↗

bioRxiv2024

Unlocking TAS2R14 activation through intricate multi-ligand binding networks

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1-3. In addition, TAS2R14 has broad expression in non-gustatory tissues, suggesting its important roles in selective physiological processes and therapeutic potential4. Here, we present cryo-electron microcopy structures of TAS2R14 in complex with flufenamic acid and aristolochic acid, coupling with G protein subtypes gustducin and Gi. Distinct from most known GPCRs, agonists of TAS2R14 bind to multiple intracellular pockets. We highlight the cholesterol molecules occupying an upper transmembrane site, typically the orthosteric pocket in other GPCRs, also binding in the entrance to an intracellular agonist binding pocket, suggesting an endogenous modulatory function5. The structural and mutagenesis analysis illuminate the receptors broad-spectrum ligand recognition and activation via intricate multiple ligand-binding sites. Furthermore, we unveil the structural configuration of gustducin and its interaction with TAS2R14, as well as the coupling mode of Gi1. This investigation should be instrumental in advancing our knowledge of the mechanisms underlying bitter taste recognition and response, with broader relevance to sensory biology and pharmacology.

biophysics↗

bioRxiv2024

Interplay of IGF1R and estrogen signaling regulates hematopoietic stem and progenitor cells

Tissue stem cells often exhibit developmental stage-specific and sexually dimorphic properties, but the underlying mechanism remains largely elusive. By characterizing IGF1R signaling in hematopoietic cells, here we report that its disruption exerts sex-specific effects in adult hematopoietic stem and progenitor cells (HSPCs). Loss of IGF1R decreases the HSPC population in females but not in males, in part due to a reduction in HSPC proliferation induced by estrogen. In addition, the adult female microenvironment enhances engraftment of wild-type but not Igf1r-null HSPCs. In contrast, during gestation, when both female and male fetuses are exposed to placental estrogens, loss of IGF1R reduces the numbers of their fetal liver HSPCs regardless of sex. Collectively, these data support the interplay of IGF1R and estrogen pathways in HSPCs and suggest that the proliferation-promoting effect of estrogen on HSPCs is in part mediated via IGF1R signaling.

cell biology↗

bioRxiv2024

Ca2+-independent ZmCPK2 is inhibited by Ca2+-dependent ZmCPK17 during drought response in maize

Calcium oscillations are induced by different stresses. Calcium-dependent protein kinases (CDPKs/CPKs) are one major group of the plant calcium decoders that are involved in various processes including drought response. Some CPKs are calcium-independent. Here, we identified ZmCPK2 as a negative regulator of drought resistance by screening an overexpression transgenic maize pool. We found that ZmCPK2 does not bind calcium, and its activity is modulated likely in an oscillation manner during short time ABA treatment. Interestingly, ZmCPK2 interacts with and is inhibited by calcium-dependent ZmCPK17, a positive regulator of drought resistance, which is activated by ABA. ZmCPK17 activity exhibits opposite patterns relative to ZmCPK2 under ABA treatment, and could prevent the nuclear localization of ZmCPK2 when co-expressed. ZmCPK2 interacts with and phosphorylates ZmYAB15, a negative transcriptional factor for drought resistance. Our results suggest that drought stress-induced Ca2+ can be decoded by corresponding CPK17 likely in an oscillation manner, thereby promoting plant adaptation to water deficit.

molecular biology↗

bioRxiv2024

Deep learning modeling of ribosome profiling reveals regulatory underpinnings of translatome and interprets disease variants

Gene expression involves transcription and translation. Despite large datasets and increasingly powerful methods devoted to calculating genetic variants effects on transcription, discrepancy between mRNA and protein levels hinders the systematic interpretation of the regulatory effects of disease-associated variants. Accurate models of the sequence determinants of translation are needed to close this gap and to interpret disease-associated variants that act on translation. Here, we present Translatomer, a multimodal transformer framework that predicts cell-type-specific translation from mRNA expression and gene sequence. We train Translatomer on 33 tissues and cell lines, and show that the inclusion of sequence substantially improves the prediction of ribosome profiling signal, indicating that Translatomer captures sequence-dependent translational regulatory information. Translatomer achieves accuracies of 0.72 to 0.80 for de novo prediction of cell-type-specific ribosome profiling. We develop an in silico mutagenesis tool to estimate mutational effects on translation and demonstrate that variants associated with translation regulation are evolutionarily constrained, both within the human population and across species. Notably, we identify cell-type-specific translational regulatory mechanisms independent of eQTLs for 3,041 non-coding and synonymous variants associated with complex diseases, including Alzheimers disease, schizophrenia, and congenital heart disease. Translatomer accurately models the genetic underpinnings of translation, bridging the gap between mRNA and protein levels, and providing valuable mechanistic insights toward mapping "missing regulation" in disease genetics.

genomics↗

bioRxiv2024

Schlafen-11 and -9 are innate immune sensors for intracellular single-stranded DNA

Nucleic acids are major structures detected by the innate immune system. Although intracellular single-stranded DNA (ssDNA) is accumulated during pathogen infection or pathogenesis, it remains unclear whether and how intracellular ssDNA stimulates the immune system. We report that intracellular ssDNA triggers cytokine expression and cell death in a CGT motif-dependent manner. Through a genome-wide CRISPRLCas9 screen, we identified Schlafen-11 (SLFN11), which is essential for the response to endogenous and pathogen ssDNA. SLFN11 directly binds ssDNA containing CGT motifs and translocates to the cytoplasm upon ssDNA recognition. The mice deficient in SLFN9, the homologue of SLFN11, were resistant to CGT ssDNA-induced inflammation, acute hepatitis and septic shock. This study establishes CGT ssDNA and SLFN11/9 as a novel type of immunostimulatory nucleic acids and pattern recognition receptors, respectively. One-sentence summariessingle-stranded DNA inside cells can awaken Schlafens with a CGT motif to trigger cytokine expression and lytic cell death.

immunology↗

bioRxiv2024

A Massively Parallel CRISPR-Based Screening Platform for Modifiers of Neuronal Activity

Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens for neuronal activity, using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2. Using this screening method, we evaluated 1343 genes for their effect on excitability in human iPSC-derived neurons, revealing potential links to neurodegenerative and neurodevelopmental disorders. These genes include known regulators of neuronal excitability, such as TARPs and ion channels, as well as genes associated with autism spectrum disorder and Alzheimers disease not previously described to affect neuronal excitability. This CRISPRi-based screening platform offers a versatile tool to uncover molecular mechanisms controlling neuronal activity in health and disease.

neuroscience↗

bioRxiv2024

Mechanical confinement induces ferroptosis through mitochondrial dysfunction

Cells exist in highly crowded environments where they are exposed to fluctuating mechanical forces arising from surrounding cells and the extracellular matrix microenvironment. In these settings, external forces are transmitted to intracellular organelles including the nucleus. While cells can survive confinement, extended duration of confinement or confinement in settings where cells are unable to escape, can result in cell death. How cells sense and respond to prolonged confinement to trigger cell death remains unclear. Here, we demonstrate that nuclear deformation generated by axial confinement triggers the programmed cell death pathway - ferroptosis. We show that axial confinement results in Drp1-dependent mitochondrial fragmentation and cPLA2 translocation to mitochondria, where Drp1 undergoes acute phase separation. Ensuing mitochondrial ROS accumulation and arachidonic acid production concertedly leads to lipid peroxidation evoking ferroptosis. Finally, of clinical relevance, we find that in human osteoarthritis tissue cPLA2 exhibits mitochondrial localization and high ROS levels. Together, our findings unveil a pivotal role for Drp1 and cPLA2 in linking mechanical confinement with mitochondrial dysfunction resulting in ferroptosis, which sheds new light on a mechanical mechanism of pathophysiology in osteoarthritis.

cell biology↗

bioRxiv2024

SlWRKY80 recruits SlHDA1 to regulates the tomato fruit ripening and color transformation

Fruit ripening involves key transcription factors (TF). Despite the established importance of WRKY TFs in plant development, the involvement of WRKY and its underlying mechanism in the regulation of fruit ripening and color transformation remain unclear. Here, we discovered a phenomenon where SlWRKY80 is involved in the ripening and color transformation of tomato. Overexpression of SlWRKY80 shortened the color transition cycle of tomato fruit, whereas loss-of-function mutation in SlWRKY80 delays this process. On the 7th day after the color breakdown (Break+7), the fruit color was bright red in SlWRKY80-overexpression lines. SlWRKY80 could bind to downstream promoters of SlSGR1 and SlPSY1, thereby exerting a positive regulatory effect on the expression levels of SlSGR1 and SlPSY1. This regulatory activity led to the facilitation of chlorophyll a decomposition and carotenoid synthesis during the transition of fruit color, ultimately accelerating fruit ripening and color change. The SlWRKY80 knockout lines (slwrky80cr-3 and slwrky80cr-4) showed light red. Thr (The 69th amino acid) mutated to Ser of SlWRKY80 (slwrky80cr-46), SlHDA1 was recruited as a regulatory factor to enhance the regulation of downstream SlPSY1 by SlWRKY80, and inhibited the SlSGR1 expression by SlWRKY80 which maybe the reason why the slwrky80cr-46 knockout lines exhibited brown color. In conclusion, SlWRKY80 regulates the expression of SlSGR1 and SlPSY1 by recruiting SlHDA1, thereby regulating chlorophyll decomposition and carotenoid synthesis, and further participating in fruit maturation and related fruit color formation. This research has important guiding significance for breeding tomato germplasm with excellent commercial traits.

plant biology↗

bioRxiv2024

Diverse competitor B cell responses to a germline-targeting priming immunogen in human Ig loci transgenic mice

Broadly neutralizing antibodies (bnAbs) have promise to protect against HIV infection, but induction of bnAbs by immunization is an unsolved vaccine design challenge. Germline-targeting priming immunogens aim to initiate the induction of bnAbs by specifically activating rare bnAb-precursor B cells that can subsequently be matured using suitable heterologous boosting and shepherding immunogens. Several pre-clinical studies, and the IAVI G001 human clinical trial, have demonstrated the ability of a germline-targeting priming immunogen, eOD-GT8 60mer, to induce precursors of the VRC01 class of bnAbs. However, much less is known about B cells induced against other epitopes of the immunogen. Here, we performed unbiased analysis of B cells induced by eOD-GT8 60mers in Intelliselect Transgenic mice (Kymice) that are transgenic for the human Ig loci and produce human-like BCRs. B cells isolated with intact eOD-GT8 60mer nanoparticles showed a large diversity of non-VRC01-class B cells, with 38% unique clonotypes and only 5% of BCRs belonging to public lineages shared among all animals. We found that many competitors recognize epitopes in close proximity to or overlapping with the VRC01 epitope. These results indicate that optimal boosting of VRC01-class bnAb-precursor B cells primed by eOD-GT8 60mer might require a first-boost immunogen that minimizes recognition of competitor B cells, and such competitors isolated from Kymice could serve as valuable reagents for boost development.

immunology↗

bioRxiv2023

Highly multiplexed, image-based pooled screens in primary cells and tissues with PerturbView

Optical pooled screening (OPS) is a highly scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture, due to limitations associated with in situ sequencing (ISS) of perturbation barcodes. Here, we developed PerturbView, an OPS technology that leverages in vitro transcription (IVT) to amplify barcodes prior to ISS, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in iPSC-derived neurons, primary immune cells, and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF{kappa}B signaling. Furthermore, we combined PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in vivo screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

systems biology↗

bioRxiv2023

Loss of Endothelial TDP-43 Leads to Blood Brain Barrier Defects in Mouse Models of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Loss of nuclear TDP-43 occurs in a wide range of neurodegenerative diseases, and specific mutations in the TARDBP gene that encodes the protein are linked to familial Frontal Temporal Lobar Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS). Although the focus has been on neuronal cell dysfunction caused by TDP-43 variants, TARDBP mRNA transcripts are expressed at similar levels in brain endothelial cells (ECs). Since increased permeability across the blood brain barrier (BBB) precedes cognitive decline, we postulated that altered functions of TDP-43 in ECs contributes to BBB dysfunction in neurodegenerative disease. To test this hypothesis, we examined EC function and BBB properties in mice with either knock-in mutations found in ALS/FTLD patients (TARDBPG348C and GRNR493X) or EC-specific deletion of TDP-43 throughout the endothelium (Cdh5(PAC)CreERT2; Tardbpff) or restricted to brain endothelium (Slco1c1(BAC)CreERT2; Tardbpff). We found that TARDBPG348Cmice exhibited increased permeability to 3kDa Texas Red dextran and NHS-biotin, relative to their littermate controls, which could be recapitulated in cultured brain ECs from these mice. Nuclear levels of TDP-43 were reducedin vitro and in vivo in ECs from TARDBPG348Cmice. This coincided with a reduction in junctional proteins VE-cadherin, claudin-5 and ZO-1 in isolated ECs, supporting a cell autonomous effect on barrier function through a loss of nuclear TDP-43. We further examined two models of Tardbp deletion in ECs, and found that the loss of TDP-43 throughout the endothelium led to systemic endothelial activation and permeability. Deletion specifically within the brain endothelium acutely increased BBB permeability, and eventually led to hallmarks of FTD, including fibrin deposition, microglial and astrocyte activation, and behavioral defects. Together, these data show that TDP-43 dysfunction specifically within brain ECs would contribute to the BBB defects observed early in the progression of ALS/FTLD.

neuroscience↗

bioRxiv2023

Scm6A: A fast and low-cost method for quantifying m6A modifications at the single-cell level

It is widely accepted that m6A exhibits significant intercellular specificity, which poses challenges for its detection using existing m6A quantitative methods. In this study, we introduce Scm6A, a machine learning-based approach for single-cell m6A quantification. Scm6A leverages input features derived from the expression levels of m6A trans regulators and cis sequence features, and found that Scm6A offers remarkable prediction efficiency and reliability. To further validate the robustness and precision of Scm6A, we applied a winscore-based m6A calculation method to conduct m6A-seq analysis on CD4+ and CD8+ T-cells isolated through magnetic-activated cell sorting (MACS). Subsequently, we employed Scm6A for analysis on the same samples. Notably, the m6A levels calculated by Scm6A exhibited a significant positive correlation with m6A quantified through m6A-seq in different cells isolated by MACS, providing compelling evidence for Scm6As reliability. We also used the scm6A-seq method to validate the reliability of our approach. Additionally, we performed single-cell level m6A analysis on lung cancer tissues as well as blood samples from COVID-19 patients, and demonstrated the landscape and regulatory mechanisms of m6A in different T-cell subtypes from these diseases. In summary, our work has yielded a novel, dependable, and accurate method for single-cell m6A detection. We are confident that Scm6A will have broad applications in the realm of m6A-related research.

bioinformatics↗