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Huang, S.

Publications and source records attributed to Huang, S..

At least 199 records · Page 11Linked to original sources
bioRxiv2024

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the protein most negatively correlated with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptors, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

cancer biology↗
bioRxiv2024

Fish Blood Response to Ash-Induced Environmental Alkalinization, and their Implications to Wildfire-Scarred Watersheds

Changes in land use, warming climate and increased drought have amplified wildfire frequency and magnitude globally. Ash mixing into aquatic systems after wildfires rapidly increases water pH, creating an additional threat to wildlife, especially species that are already threatened, endangered and/or migratory. Here, Chinook salmon (Oncorhynchus tshawytscha) yearlings acclimated to 15 or 20{degrees}C were exposed to an environmentally relevant concentration of ash (0.25% w/v) which caused water pH to rapidly rise from [~]8.1 to [~]9.2. Mortalities occurred within the first 12 hours, and was higher at the higher temperature (33 versus 20 %). The greatest differences in blood chemistry between the two temperatures were dramatically greater ([~]7.5-fold) and very rapid (within 1 hour) spikes in both plasma total ammonia (to [~]1200 {micro}M) and lactate (to [~]6 mM) in warm-acclimated salmon, whereas cold-acclimated salmon experienced a much smaller and gradual rise in plasma total ammonia. Salmon at both temperatures experienced extracellular and intracellular alkalosis within 1 hour that recovered within 24 hours, but the alkalosis was smaller in magnitude in fish at warmer temperature. Impacts on plasma ion concentrations were relatively mild and plasma glucose increased by 2- to 4-fold at both temperatures. Notably, the increase in plasma total ammonia in fish at the warmer temperature was far faster and much greater than those reported in previous studies exposing fish despite higher water pH (9.4-10.5) induced without using ash. This suggests that ash has physiological impacts that cannot be explained by high water pH alone which may relate to the complex mixture of metals and organic compounds also released from ash. This demonstrates post-wildfire ash input can induce lethal yet previously unexplored physiological disturbances in fish and highlights the complex interaction with warmer temperatures typical of wildfire-scarred landscapes.

physiology↗
bioRxiv2024

Multi-omic characterization of allele-specific regulatory variation in hybrid pigs

Genetic variation in the regulation of gene expression contributes substantially to phenotypic variation. Understanding how variation in DNA sequences and epigentic modifications leads to gene expression variation remains a challenging task. In hybrid animals where cellular environments are homogeneous, differences between the expression of paternal and maternal alleles must be due to cis sequence or epigenic differences. Therefore, hybrid mapping is a powerful approach to efficiently identify and characterize genes under regulation through mechanisms in cis. In this study, using reciprocal crosses of the phenotypically divergent Duroc and Lulai pig breeds, we performed a comprehensive multi-omic characterization of regulatory variation across brain, liver, muscle, and placenta in four developmental stages. We produced one of the largest multi-omic datasets to date in pigs, including 16 whole genome sequenced genomes, 48 whole genome bisulfite sequencing, 168 ATAC-Seq and 168 RNA-Seq samples. We developed a novel read count-based method to reliably assess allele-specific methylation, chromatin accessibility, and RNA expression. We showed that tissue specificity was much stronger than developmental stage specificity in all of DNA methylation, chromatin accessibility, and gene expression. We identified 573 genes showing allele specific expression, including those influenced by parent-of-origin as well as allele genotype effects. By integrating methylation, chromatin accessibility, and gene expression data, many of these allele specific expression can be explained by allele specific methylation and/or chromatin accessibility. This study provides one of the most comprehensive characterizations of regulatory variation across multiple tissues and developmental stages in pigs and offers new opportunity of genetic improvement for this important food animal species.

developmental biology↗
bioRxiv2023

AlphaFold2 structures template ligand discovery

AlphaFold2 (AF2) and RosettaFold have greatly expanded the number of structures available for structure-based ligand discovery, even though retrospective studies have cast doubt on their direct usefulness for that goal. Here, we tested unrefined AF2 models prospectively, comparing experimental hit-rates and affinities from large library docking against AF2 models vs the same screens targeting experimental structures of the same receptors. In retrospective docking screens against the {sigma}2 and the 5-HT2A receptors, the AF2 structures struggled to recapitulate ligands that we had previously found docking against the receptors experimental structures, consistent with published results. Prospective large library docking against the AF2 models, however, yielded similar hit rates for both receptors versus docking against experimentally-derived structures; hundreds of molecules were prioritized and tested against each model and each structure of each receptor. The success of the AF2 models was achieved despite differences in orthosteric pocket residue conformations for both targets versus the experimental structures. Intriguingly, against the 5-HT2A receptor the most potent, subtype-selective agonists were discovered via docking against the AF2 model, not the experimental structure. To understand this from a molecular perspective, a cryoEM structure was determined for one of the more potent and selective ligands to emerge from docking against the AF2 model of the 5-HT2A receptor. Our findings suggest that AF2 models may sample conformations that are relevant for ligand discovery, much extending the domain of applicability of structure-based ligand discovery.

pharmacology and toxicology↗
bioRxiv2023

Liver Protects Cytoarchitecture, Neuron Viability and Electrocortical Activity in Post-Cardiac Arrest Brain Injury

BACKGROUNDBrain injury is the major reason for patient deaths in victims who survive after cardiac arrest. Clinical studies have shown that the presence of hypoxic hepatitis and pre-cardiac arrest liver disease is associated with increased mortality and inferior neurological recovery. However, how the liver might impact the pathogenesis of post-cardiac arrest is still unknown. METHODSAn in vivo global cerebral ischemia model was established to assess how simultaneous liver ischemia affected the recovery of brain ischemic injury. In addition, an ex vivo brain normothermic machine perfusion (NMP) model was established to evaluate how addition of a functioning liver might impact the circulation, cytoarchitecture, neuron viability and electrocortical activity of the reperfused brain post-cardiac arrest. RESULTSIn the in vivo model, we observed a larger infarct area in the frontal lobe, elevated tissue injury scores in the CA1 region, as well as increased intravascular immune cell adhesion in the reperfused brains with hepatic ischemia, compared to those without simultaneous hepatic ischemia. The results of the ex vivo model demonstrated that the addition of a functioning liver to the brain NMP circuit significantly reduced post-cardiac arrest brain injury, increased neuronal viability and improved electrocortical activity. Furthermore, we observed significant alterations in gene expressions and metabolites in the presence or absence of hepatic ischemia. CONCLUSIONSOur research highlights the crucial role of the liver in the pathogenesis of post-cardiac arrest brain injury. These findings shed lights on a cardio-pulmonary-hepatic brain resuscitation strategy for patients with cardiac arrest.

neuroscience↗
bioRxiv2023

Utilizing Non-Invasive Prenatal Test Sequencing Data Resource for Human Genetic Investigation

Non-invasive prenatal testing (NIPT) employs ultra-low-pass sequencing of maternal plasma cell-free DNA to detect fetal trisomy. With exceptional sensitivity, specificity, and safety, NIPT has gained global adoption, exceeding ten million tests, establishing it as one of the largest human genetic resources. This resource holds immense potential for exploring population genetic variations and their correlations with phenotypes. Here, we present comprehensive methods tailored for analyzing large, low-depth NIPT genetic datasets, involving customized algorithms and software for genetic variation detection, genotype imputation, and genome-wide association analysis. Through evaluations, we demonstrate that, when integrated with appropriate probabilistic models and population-specific haplotype reference panels, accurate allele frequency estimation and high genotype imputation accuracy (0.8 to 0.9) are achievable for genetic variants with alternative allele frequencies between 0.01 and 0.05, at sequencing depths of 0.1x to 0.25x. Additionally, we attained an R-square exceeding 0.9 for estimating genetic effect sizes across various sequencing platforms. These findings establish a robust theoretical and practical foundation for leveraging NIPT data in advancing medical genetic studies, not only in realms of maternal and child health, but also for long-term health outcomes. HighlightsO_LIIntroduction of probabilistic model integration for analyzing large-scale, low-pass non-invasive prenatal test (NIPT) sequencing data C_LIO_LIEvaluation of protocols for variant detection, genotype imputation, and genome-wide association analyses with NIPT data C_LI

bioinformatics↗
bioRxiv2023

Familiarity training enhance straightening of neural trajectory for video prediction

Predictive processing in the visual system is pivotal for efficient sensory-driven behaviors. Previous research has shown that the visual system transforms sequential inputs into straighter temporal trajectories. However, the specific role of neural straightening in predictive processing, especially in how learning reshapes this phenomenon for enhanced prediction, remains unclear. To address this, we analyzed V2 population activity in macaques during familiarity training with video stimuli. Our findings reveal that repeated exposure to the same movies significantly enhances neural straightening, indicating a critical role of learning in refining neural trajectories for prediction. In parallel, our studies with the deep predictive network model, Pred-Net, demonstrated similar enhancements in neural straightening in response to familiar movies. This underscores a strong association between neural straightening and predictive coding. Together, our results provide novel insights into the adaptive mechanisms of the visual cortex, enriching our understanding of how learning shapes neural path-ways for efficient prediction.

neuroscience↗
bioRxiv2023

CK-666 and CK-869 differentially inhibit Arp2/3 iso-complexes

The inhibitors, CK-666 and CK-869, are widely used to probe the function of actin nucleation by the Arp2/3 complex in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 is also only able to inhibit Arp3-but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.

biochemistry↗
bioRxiv2023

Well-Paired-Seq2: High-Throughput and High-Sensitivity Strategy for Characterizing Low RNA-Content Cell/Nucleus Transcriptomes

High-throughput single-cell RNA sequencing (scRNA-seq) is recognized as a powerful technology for disentangling the heterogeneity of cellular states. However, the Poisson-dependent cell capture and low sensitivity in scRNA-seq methods pose challenges for throughput and for samples with low RNA-content. Herein, to address these challenges, we developed Well-Paired-Seq2 (WPS2) based on size-exclusion and locally quasi-static hydrodynamic principles to realize high efficiency of cell utilization, single cell/bead pairing, and cell-free RNA removal. WPS2 exploits molecular crowding effect, tailing activity enhancement in reverse transcription, and homogeneous enzymatic reaction in the initial bead-based amplification to achieve 3116 genes and 8447 transcripts. With an average of [~]20,000 reads per cell, WPS2 detected 1420 more genes and 4864 more transcripts than our previous Well-Paired-Seq. Using WPS2, we overcame the Poisson limit for the capture of both cells and beads and accurately characterized transcriptomes of low RNA-content single cells and nuclei with high sensitivity. WPS2 was further applied to comprehensively profile transcriptomes from frozen clinical samples. We found that clear cell renal cell carcinoma (ccRCC) has a complex microenvironment, and that chromophobe renal cell carcinoma (chRCC) exhibits abundant copy number variations (CNVs). In addition, metanephric adenoma (MA) was characterized at single-cell level for the first time and some potentially specific markers were revealed. With the advantages of high sensitivity, high throughput, and high fidelity, we anticipate that WPS2 will be broadly applicable in basic and clinical research.

genetics↗
bioRxiv2023

Positive selection analyses identify a single WWE domain residue that shapes ZAP into a super restriction factor

The host interferon pathway upregulates intrinsic restriction factors in response to viral infection. Many of them block a diverse range of viruses, suggesting that their antiviral functions might have been shaped by multiple viral families during evolution. Virus-host conflicts have led to the rapid adaptation of viral and host proteins at their interaction hotspots. Hence, we can use evolutionary genetic analyses to elucidate antiviral mechanisms and domain functions of restriction factors. Zinc finger antiviral protein (ZAP) is a restriction factor against RNA viruses such as alphaviruses, in addition to other RNA, retro-, and DNA viruses, yet its precise antiviral mechanism is not fully characterized. Previously, an analysis of 13 primate ZAP identified 3 positively selected residues in the poly(ADP-ribose) polymerase-like domain. However, selective pressure from ancient alphaviruses and others likely drove ZAP adaptation in a wider representation of mammals. We performed positive selection analyses in 261 mammalian ZAP using more robust methods with complementary strengths and identified 7 positively selected sites in all domains of the protein. We generated ZAP inducible cell lines in which the positively selected residues of ZAP are mutated and tested their effects on alphavirus replication and known ZAP activities. Interestingly, the mutant in the second WWE domain of ZAP (N658A) is dramatically better than wild-type ZAP at blocking replication of Sindbis virus and other ZAP-sensitive alphaviruses due to enhanced viral translation inhibition. The N658A mutant inhabits the space surrounding the previously reported poly(ADP-ribose) (PAR) binding pocket, but surprisingly has reduced binding to PAR. In summary, the second WWE domain is critical for engineering a super restrictor ZAP and fluctuations in PAR binding modulate ZAP antiviral activity. Our study has the potential to unravel the role of ADP-ribosylation in the host innate immune defense and viral evolutionary strategies that antagonize this post-translational modification. Author summaryHost proteins and viral proteins that encounter one another are locked in a perpetual genetic arms race. In this evolutionary race, a mutation that confers a survival advantage will become more frequent in the population. By looking at the sequences of genes that are known to have antiviral roles in mammals, we can identify the exact sites where a host and viral protein have interacted and gain insight into how an antiviral protein works. Here, we identified these sites in zinc finger antiviral protein (ZAP), a host protein that blocks many different viruses. We found that changing one of the sites from the original amino acid to another dramatically improves ZAPs antiviral activity against Sindbis virus, an alphavirus, due to improved inhibition of viral translation. Our mutation is also better at inhibiting other members in the Alphavirus genus. We observed that our mutant ZAP has reduced ability to bind poly(ADP-ribose), a post-translational modification that is targeted by alphaviruses for productive infection. Our findings help us better understand how viruses have shaped the evolution of broad-spectrum host antiviral proteins, with great implications for the engineering of super restriction factors.

microbiology↗
bioRxiv2023

Examining models of modern human origins through the analysis of 43 fully sequenced human Y chromosomes

Molecular studies have yielded two primary models for understanding the uniparental DNA phylogenetic trees of modern humans: the Recent Out of Africa (ROA) and the Recent Out of East Asia (ROE) models. These models differ in their underlying assumptions, particularly in relation to early stem haplotypes, even though they share many haplotype relationships. Leveraging the wealth of new genetic variants within the male-specific region unveiled through the comprehensive sequencing of 43 diverse human Y chromosomes, we here investigated the presence of shared variants among different haplotypes to determine which model better aligns with the genetic data. We were able to corroborate the existence of stem haplotypes specific to the ROE model, but not those exclusive to the ROA model. We found that A0b and A1a shared the most variants with each other, aligning with the A00A1a clade of the ROE model. Also, stem haplotypes specific to the ROE model showed the expected relationships, with A00A1a closest to B, AB closest to E, E closest to B, A, and C, ABDE closest to C. Our findings also revealed extensive variant sharing independent of common ancestry, consistent with the maximum genetic diversity theory underlying the ROE model but challenging the neutral theory behind the ROA model. These new genetic data lend robust support to the ROE model as the more accurate representation of modern human origins.

evolutionary biology↗
bioRxiv2023

Coordinated differentiation of human intestinal organoids with functional enteric neurons and vasculature

Human intestinal organoids (HIOs) derived from human pluripotent stem cells co-differentiate both epithelial and mesenchymal lineages in vitro but lack important cell types such as neurons, endothelial cells, and smooth muscle. Here, we report an in vitro method to derive HIOs with epithelium, mesenchyme, enteric neuroglial populations, endothelial cells, and organized smooth muscle in a single differentiation, without the need for co-culture. When transplanted into a murine host, these populations expand and organize to support organoid maturation and function. Functional experiments demonstrate enteric nervous system function, with HIOs undergoing peristaltic-like contractions, suggesting the development of a functional neuromuscular unit. HIOs also form functional vasculature, demonstrated in vitro using microfluidic devices to introduce vascular-like flow, and in vivo following transplantation, where HIO endothelial cells anastomose with host vasculature. Collectively, we report an in vitro model of the human gut that simultaneously co-differentiates epithelial, stromal, endothelial, neural, and organized muscle populations.

bioengineering↗
bioRxiv2023

Fourth dose of Microneedle Array Patch of SARS-CoV-2 S1 Protein Subunit Vaccine Elicits Robust Long-lasting Humoral Responses in mice

The COVID-19 pandemic has underscored the pressing need for safe and effective booster vaccines, particularly in considering the emergence of new SARS-CoV-2 variants and addressing vaccine distribution inequalities. Dissolving microneedle array patches (MAP) offer a promising delivery method, enhancing immunogenicity and improving accessibility through the skins immune potential. In this study, we evaluated a microneedle array patch-based S1 subunit protein COVID-19 vaccine candidate, which comprised a bivalent formulation targeting the Wuhan and Beta variant alongside a monovalent Delta variant spike proteins in a murine model. Notably, the second boost of homologous bivalent MAP-S1(WU+Beta) induced a 15.7-fold increase in IgG endpoint titer, while the third boost of heterologous MAP-S1RS09Delta yielded a more modest 1.6-fold increase. Importantly, this study demonstrated that the administration of four doses of the MAP vaccine induced robust and long-lasting immune responses, persisting for at least 80 weeks. These immune responses encompassed various IgG isotypes and remained statistically significant for one year. Furthermore, neutralizing antibodies against multiple SARS-CoV-2 variants were generated, with comparable responses observed against the Omicron variant. Overall, these findings emphasize the potential of MAP-based vaccines as a promising strategy to combat the evolving landscape of COVID-19 and to deliver a safe and effective booster vaccine worldwide.

immunology↗
bioRxiv2023

Probiotic neoantigen delivery vectors for precision cancer immunotherapy

Microbial systems have been synthetically engineered to deploy therapeutic payloads in vivo1-4. With emerging evidence that bacteria naturally home to tumors5-7 and modulate anti-tumor immunity8,9, one promising application is the development of bacterial vectors as precision cancer vaccines10-12. In this study, we engineered probiotic E. coli Nissle 1917 (EcN) as an anti-tumor vaccination platform optimized for enhanced production and cytosolic delivery of neoepitope-containing peptide arrays, with increased susceptibility to blood clearance and phagocytosis. These features enhance both safety and immunogenicity, achieving a system which drives potent and specific T cell-mediated anti-cancer immunity that effectively controls or eliminates tumor growth and extends survival in advanced murine primary and metastatic solid tumors. We demonstrate that the elicited anti-tumor immune response involves extensive priming and activation of neoantigen-specific CD4+ and CD8+ T cells, broader activation of both T and NK cells, and a reduction of tumor-infiltrating immunosuppressive myeloid and regulatory T and B cell populations. Taken together, this work leverages the advantages of living medicines to deliver arrays of tumor-specific neoantigen-derived epitopes within the optimal context to induce specific, effective, and durable systemic anti-tumor immunity.

synthetic biology↗
bioRxiv2023

Reconciling ASPP-p53 Binding Mode Discrepancies through an Ensemble Binding Framework that Bridges Crystallography and NMR Data

ASPP2 and iASPP bind to p53 through their conserved ANK-SH3 domains to respectively promote and inhibit p53-dependent cell apoptosis. While crystallography has indicated that these two proteins employ distinct surfaces of their ANK-SH3 domains to bind to p53, solution NMR data has suggested similar surfaces. In this study, we employed multi-scale molecular dynamics (MD) combined with free energy calculations to reconcile the discrepancy in the binding modes. We demonstrated that the binding mode based solely on a single crystal structure does not enable iASPPs RT loop to engage with p53s C-terminal linker--a verified interaction. Instead, an ensemble of simulated iASPP-p53 complexes facilitates this interaction. We showed that the ensemble-average inter-protein contacting residues and NMR-detected interfacial residues align well with ASPP proteins, and the ensemble-average binding free energies better match experimental Kd values compared to single crystallgarphy-determined binding mode. For iASPP, the sampled ensemble complexes can be grouped into two classes, resembling the binding modes determined by crystallography and solution NMR. We thus propose that crystal packing shifts the equilibrium of binding modes towards the crystallographydetermined one. Lastly, we show that the ensemble binding complexes are sensitive to p53s intrinsically disordered regions (IDRs), attesting to experimental observations that these IDRs contribute to biological functions. Our results provide a dynamic and ensemble perspective for scrutinizing these important cancer-related protein-protein interactions (PPIs).

biophysics↗
bioRxiv2023

Parkinson's genes orchestrate pyroptosis through selective trafficking of mtDNA to leaky lysosomes

Inflammation is an age-related factor that underlies numerous human disorders. A key driver of inflammation is the release of mitochondrial DNA (mtDNA), which binds and activates cytosolic sensors. This induces transcriptional responses and, ultimately, pyroptotic cell death. The main challenge has been to understand how mtDNA can cross the two mitochondrial membranes to access the cytosol. Through a genome-wide CRISPR knockout screen we identified a new pyroptotic pathway defined by mtDNA exit within mitochondrial-derived vesicles that are delivered to lysosomes. Critically, breach of lysosomes allows mtDNA to access cytosol, requiring multiple Parkinsons Disease-related proteins and Gasdermin pores, identified in the screen. These data place mitochondria-to-lysosome transport as a driver of pyroptosis and link multiple PD proteins along a common pathway. One sentence summaryParkinsons disease-related proteins regulate pyroptosis

cell biology↗
bioRxiv2023

SOD1 is a synthetic lethal target in PPM1D-mutant leukemia cells

The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase, Mg2+/Mn2+ dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacologic target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Altogether, our results demonstrate the protective role of SOD1 against oxidative stress in PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

cancer biology↗