Biology subjects

Huang, P.

Publications and source records attributed to Huang, P..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Functional impact of PCSK9 variants on LDL uptake in a knockout hepatic model

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a central regulator of low-density lipoprotein (LDL) cholesterol metabolism, yet the functional consequences of many clinically observed PCSK9 variants remain unknown. To establish a rigorous system for quantitative variant assessment, we generated a PCSK9 knockout (KO) HepG2 cell line through CRISPR/Cas9-mediated deletion of exons 2-8, effectively removing both the prodomain and catalytic regions required for PCSK9 function. This null background enabled systematic functional mapping of wild-type (WT) PCSK9 and multiple clinically relevant variants representing well-characterized, recurrent, and previously understudied alleles. Functional assays revealed pronounced heterogeneity among variant activities. The classical gain-of-function (GOF) variants D374Y and R496W exhibited robust suppression of LDL uptake, whereas A443T--an infrequently reported and previously uncharacterized variant--demonstrated a loss-of-function (LOF)-like phenotype with significantly enhanced LDL uptake. Additional poorly characterized variants, including V4I, R104C/V114A, and R496W/N425S, displayed minimal functional profiles, providing novel mechanistic insights. Surface LDL receptor (LDLR) levels generally correlated with LDL uptake but revealed unique patterns for specific variants. This KO-based rescue system provides a high-resolution framework for mechanistic classification of both established and poorly characterized PCSK9 variants, bridging the gap between genetic discovery and functional interpretation while supporting precision lipid-lowering strategies.

biochemistry↗

bioRxiv2026

3D, multi-omic imaging reveals molecular biomarkers of the pre-metastatic niche in lung cancer

The recurrence rate following complete surgical resection of primary non-small cell lung cancer is as high as 55%, yet no approach currently exists to evaluate the risk of local recurrence. The premetastatic paradigm is the recognition that metastasis is preceded by reprogramming naive tissues to prime a microenvironment for tumor cell survival and subsequent reactivation. Identification of biomarkers of the pre-metastatic niche would allow us to evaluate a patients risk of local relapse in the normal lung parenchyma surrounding the resected tumor. We designed a workflow incorporating in vivo modelling, radiology, and deep learning-guided three-dimensional (3D) imaging, spatial proteomics, and transcriptomics to identify previously unreported signals associated with the early transformation of the lung parenchyma announcing regional metastasis. We curated biorepository spanning timepoints before and after resection of primary Lewis Lung Carcinoma (LLC) tumors. Using radiology and cellular resolution 3D histology, we calculated the number and distribution of metastases in mouse lungs and developed an algorithm to guide placement of spatial proteomics and transcriptomics to regions containing early micro-metastases and the pre-metastatic microenvironment. Molecular and tissue features associated with presence, size, and location of metastases guided the identification of both myeloid (F4/80) and senescent (p16/p21) cell signatures in the premetastatic and metastatic environments. Finally, multiparametric flow cytometry of metastatic lungs in a senescence reporter GEMM (tdTomato-p16 INKA mice) resolved senescent cells including alveolar macrophages as the cellular phenotypes associated with these early premetastatic signatures. Altogether, this work highlights a novel AI-assisted approach for detection of biomarkers of tissue remodeling during lung cancer invasion.

bioengineering↗

bioRxiv2026

Chronic Paraventricular OX1R Overexpression Induces Oxidative Stress and Hypertension in Rats

BackgroundHyperactivity of the orexin system has been implicated in hypertension, yet the long-term impact of orexin 1 receptor (OX1R) overexpression in the paraventricular nucleus (PVN), a key brain region involved in arterial blood pressure (ABP) regulation, remains unclear. This study examined whether chronic OX1R overexpression in the PVN alters cardiovascular, neuroendocrine, and oxidative functions. MethodsAdult male Sprague-Dawley rats received bilateral PVN injections of AAV2-OX1R or control virus AAV2-GFP. Their ABP and heart rate (HR) were monitored for eight weeks via radiotelemetry. Renal sympathetic nerve activity (RSNA) and ABP responses to PVN orexin A administration were assessed using in vivo recordings. Reactive oxygen species (ROS) were quantified using MitoProbe, and gene expression was analyzed by qPCR. Primary neuronal cultures were used to explore underlying mechanism involved in increased ROS production and neuronal activity. ResultsPVN OX1R overexpression increased mean ABP ([~]10 mmHg), water intake, and ROS levels in both the PVN and peripheral organs. In addition, orexin A evoked exaggerated RSNA (155% vs. 73%) and pressor responses (27 vs. 16 mmHg) in PVN OX1R overexpression rats compared to control rats, these effects were attenuated by the OX1R antagonist SB408124. Plasma vasopressin levels were elevated, and brain sections confirmed increased ROS in OX1R-positive neurons. In vitro experiments further demonstrated that orexin A upregulated ROS-generating enzymes, suppressed antioxidant genes, and enhanced neuronal excitability. ConclusionChronic PVN OX1R overexpression induces oxidative stress, sympathetic overactivity, and increased plasma vasopressin, contributing to hypertension development. Targeting PVN OX1R signaling may provide therapeutic benefit for hypertension.

physiology↗

bioRxiv2025

B Cell-Induced Lymph Node Stromal Remodeling Compromises Neutrophil Response to Secondary Staphylococcus aureus Infection

The lymph node (LN) stromal cells create niches, regulate lymph drainage, and guide the migration, positioning, and activation of immune cells within the LN. Upon stimulation, the stromal cells respond dynamically to accommodate the massive expansion of immune cells in the LN. However, there is still a limited understanding of how niche-associated stromal cell subsets are remodeled and how this remodeling reshapes host immune protection. In an oxazolone (OX)-induced skin inflammation model, we found that LN remodeling weakened the neutrophil response to secondary Staphylococcus aureus (S. aureus) infection in the inflamed LN, whereas depleting B cells rescued the responses. To understand the mechanism, we used single-cell RNA sequencing (scRNAseq) to characterize alterations in fibroblastic reticular cell (FRC) subsets and identified a new FRC subset expressing an intermediate level of CXCL13 (Cxcl13int RCs) in the inflamed LN. Further studies revealed that Cxcl13int RCs replaced Ccl19lo FRCs in the interfollicular zones (IFZs) of the inflamed LN, leading to a compromised conduit network. Depleting B cells preserved the integrity and function of the FRC-conduit network within the IFZ. Finally, the induction of Cxcl13int RCs depended on the B cell-derived lymphotoxin signaling. The induced Cxcl13int RCs were transitional, creating a time window of comprised immune protection during the OX-inflammation. This study elucidated a mechanism underlying LN stromal remodeling and provides proof-of-principle evidence that B cell-stromal interactions may be a novel target to enhance host immunity in diseased conditions.

immunology↗

bioRxiv2025

ADAPT-M: A workflow for rapid, quantitative in vitro measurements of enriched protein libraries

Protein-protein interactions underpin most cellular interactions, and engineered binders present powerful tools for probing biology and developing novel therapeutics. One bottleneck in binder generation is the scalable, quantitative characterization of these interactions. We present ADAPT-M (Affinity Determination by Adaptation of ProTein binders for Microfluidics), a streamlined workflow that connects yeast surface display (YSD) with in vitro affinity and kinetic measurements using the high-throughput STAMMPPING microfluidic platform. ADAPT-M quantifies Kds and dissociation kinetic parameters for hundreds of enriched protein variants in under one week without requiring hands-on protein purification. We applied ADAPT-M to a computationally designed library targeting the SARS-CoV-2 Omicron BA.1 receptor binding domain, successfully recovering and measuring Kds for most highly enriched YSD variants. Measurements correlate strongly with biolayer interferometry and yeast titration assays. ADAPT-M further enabled selection of lead candidates for structural and mutational analysis, which revealed designed paratopes were preserved despite binding to off-target epitopes. By bridging YSD screening and in vitro validation, ADAPT-M accelerates protein binder discovery and supports data-driven protein engineering.

biophysics↗

bioRxiv2025

Genomic and genetic insights into speciation and pigment pattern diversification in Danio fishes

The Danioninae subfamily of teleost fishes boasts up to four hundred distinct species that have evolved to display a stunning diversity of morphological forms. Here we use newly assembled genome sequences of four laboratory and wild zebrafish strains as well as eleven species of the Danio and Danionella genera to explore their phylogenetic history and the genetic basis of pigment pattern diversification. Phylogenomic analyses uncover extensive introgression and incomplete lineage sorting that have obscured phylogenetic relationships within Danio and corroborate an ancient hybrid origin of zebrafish. Whereas D. rerio inherited ancestral horizontal stripes, relatives repeatedly evolved spots and vertical bars. Interspecific complementation tests reveal functional divergence of the adhesion molecule gene igsf11 and the gap junction gene gja5b between the striped zebrafish and Danio species with divergent patterns. Comparative genomic and transcriptomic analyses suggest that protein and regulatory evolution have accompanied pigment pattern diversification. Our analyses elucidate complex genetic changes underlying the phylogenetic history and morphological diversification in the Danio genus. Resolved phylogenetic relationships, available genome assemblies, transcriptomes, and genetic tractability establish Danio fish species as excellent models for biomedical research in vertebrates.

genomics↗

bioRxiv2025

Schistosoma japonicum Histone Acetyltransferase 1 (SjHAT1): A Novel Anti-schistosomal Drug Target

Schistosomiasis remains a critical global health issue, necessitating novel therapies due to emerging praziquantel resistance. We previously developed a patented praziquantel derivative, DW-3-15, which demonstrated potent broad-spectrum schistosomicidal activity through SjHAT1 inhibition. Here, the full-length SjHAT1 cDNA is cloned by rapid amplification of cDNA ends methods. The amino acids encoded by the cDNA retains conserved catalytic residues of acetyltransferases although sharing only 34% identity with mammalian orthologs. Phylogenetic analyses place it in a distinct clade, indicating its divergence specific to Schistosoma species. The expression profiling of SjHAT1 reveals stage- and sex-specific patterns. Fluorescence in situ hybridization localizes SjHAT1 predominantly in female vitellaria and male parenchyma near the gynecophoral canal. Knockdown of SjHAT1 impairs worm survival, reduces female oviposition and disrupts ovarian and vitelline morphology both in vitro and in vivo. RNA-sequencing analysis reveals that SjHAT1 knockdown disrupts {beta}-alanyl-tryptamine pheromone signaling via downregulation of aromatic L-amino acid decarboxylase in males and multidrug resistance-associated protein 4 in females. Through exploring this dual-sex regulatory mechanisms, SjHAT1 may emerge as a promising therapeutic target for interrupting schistosomiasis transmission.

microbiology↗

bioRxiv2025

Leveraging protein representations to explore uncharted fold spaces with generative models

A major challenge in computational de novo protein design is the exploration of uncharted areas within the protein structural space. However, the large degrees of freedom of protein backbones complicate the sampling process during protein design. Machine learning-based models have made great strides in this problem, however due to their nature they tend to exploit rather than explore the data distribution used for training the neural networks. To address some of these challenges, we propose a new coarse grained protein structure representation generative method, DiffTopo, a diffusion model which increases the sampling efficiency and diversity. Combined with a backbone level protein generative model like RFdiffusion, novel protein folds can be generated rapidly, allowing for efficient exploration of the designable topology space. Interestingly, we have discovered that by mirroring the topological organization of native proteins using a pipeline named MirrorTopo, we can readily expand the known fold space. We generated and experimentally characterized 30 different novel topologies from DiffTopo and 6 different novel mirror topologies from MirrorTopo. The developed framework relying on low resolution sampling provides new means for fold exploration challenges, which could in principle enhance our knowledge of the first principles of protein structure and folding, as well as create new opportunities for functional design.

bioinformatics↗

bioRxiv2025

SLAE: Strictly Local All-atom Environment for Protein Representation

AO_SCPLOWBSTRACTC_SCPLOWBuilding physically grounded protein representations is central to computational biology, yet most existing approaches rely on sequence-pretrained language models or backbone-only graphs that overlook side-chain geometry and chemical detail. We present SLAE, a unified all-atom framework for learning protein representations from each residues local atomic neighborhood using only atom types and interatomic geometries. To encourage expressive feature extraction, we introduce a novel multi-task autoencoder objective that combines coordinate reconstruction, sequence recovery, and energy regression. SLAE reconstructs allatom structures with high fidelity from latent residue environments and achieves state-of-the-art performance across diverse downstream tasks via transfer learning. SLAEs latent space is chemically informative and environmentally sensitive, enabling quantitative assessment of structural qualities and smooth interpolation between conformations at all-atom resolution.

bioinformatics↗

bioRxiv2025

Lysine-R2HGylation identified as a post-translational modification in R2HG-elevated cancers

R-2-Hydroxyglutarate (R2HG), an oncometabolite predominantly produced by mutated isocitrate dehydrogenase 1/2 (IDH1/2) in various cancers, is known to drive cancer progression through noncovalent inhibition of -ketoglutarate (KG)-dependent enzymes. In this work, we propose an alternative mechanism wherein R2HG contributes to cancer development via covalent modification of biologically critical lysines, a process termed lysine-R2HGylation (KR2HG). We designed and synthesized R2HG-mimicking probes, demonstrating their effectiveness in facilitating KR2HG target profiling and site mapping. We identified KR2HG as a previously unrecognized post-translational modification, confirmed its C5-linkage on GSTP1(K209), and demonstrated that SIRT5 functions as a deacylase for GSTP1-KR2HG in vitro. Furthermore, we found that R2HG slightly but significantly inhibits the enzymatic activity of GSTP1 through KR2HG and dramatically suppresses monocyte differentiation via this catalytically important lysine modification. Our findings provide an alternative perspective on the role of R2HG in leukemia progression and offer a practical tool for the clinical investigation of R2HG-elevated cancers.

molecular biology↗

bioRxiv2025

Ensemble-conditioned protein sequence design with Caliby

Structure-conditioned sequence design models aim to design a protein sequence that will fold into a given target structure. Deep-learning-based approaches for sequence design have proven highly successful for various protein design applications, but many non-idealized backbones still remain out of reach for current models under typical in silico success criteria. We hypothesize that training objectives prioritizing native sequence recovery unintentionally push models to reproduce non-structural signals (e.g. phylogenetic relatedness, neutral drift, or dataset sampling biases), rather than a broadly generalizable structure-sequence mapping. Inspired by recent work bridging sequence likelihood and fitness prediction in protein language models, we introduce Caliby, a Potts model-based sequence design method capable of conditioning on an ensemble of structures. Conditioning on a synthetic ensemble generated from an input backbone allows sampling of sequences consistent with the structural constraints of the ensemble while averaging out undesired biases towards the native sequence. Ensemble-conditioned sequence design with Caliby reduces native sequence recovery while substantially improving AlphaFold2 self-consistency, outperforming state-of-the-art models ProteinMPNN and ChromaDesign on both native and de novo backbones. Finally, we train a variant of Caliby on only soluble proteins and demonstrate in silico that Protpardelle-1c binder designs that were previously deemed undesignable by SolubleMPNN are actually designable under SolubleCaliby, highlighting limitations of existing filtering pipelines. These results suggest that Caliby can expand the de novo design space beyond highly idealized backbones.

bioengineering↗

bioRxiv2025

The anti-phage mechanism of a widespread trypsin-MBL module

Protease mediate activation of immune effectors is a conserved mechanism across life. This study identified a widespread and co-evolving trypsin-MBL module as a core effector in diverse bacterial immune systems (e.g., Hachiman, Argonaute, AVAST), protecting host via abortive infection. Focusing on Hachiman-trypsin-MBL, we demonstrated that, the protease activity of trypsin*HamAB is inhibited in the presence of ATP and MBL is an autoinhibited DNase with two inserted loops blocking the catalytic pocket. Upon infection, the HamB helicase senses invading nucleic acids and catalyzes the hydrolysis of ATP, activating the associated trypsin-like protease that cleaves specifically at two non-canonical KSS motif of MBL and releases its DNase activity. Cryo-EM structures of activated trypsin*HamAB bound with DNA reveal DNA binding and ATP hydrolysis lead to the release of the trypsin-like domain, enabling activation. Our work provides a comprehensive understanding on the mechanism of widely distributed trypsin-MBL module, wherein proteolytic activation of a toxic nuclease enables robust bacterial immunity while preventing self-toxicity through multi-layered control.

biochemistry↗

bioRxiv2025

Engineered MS2 Virus Capsids for Cellular Display of Peptide Antigens

Our ability to respond to emerging pandemics and pathogen resistance relies critically on our ability to build vaccines quickly and efficiently. In this report we used an efficient enzymatic oxidative coupling reaction to create a viral capsid-based vaccine platform that is modular and quickly adaptable for many different pathogens. Tyrosinase-mediated oxidative coupling was used to conjugate C-terminal tyrosine residues on peptide antigens to cysteine residues installed inside MS2 viral capsids. This strategy is particularly promising because the capsids protect the internally conjugated peptides from protease degradation before they are delivered into cells. The vaccine constructs were tested for MHC presentation followed by T-cell activation. Mutants of the MS2 capsid itself activated DC2.4 cells, serving as an adjuvant to help induce the immune response to delivered antigens. The MS2-peptide constructs were shown to be stable in serum, activate DC2.4 cells, and lead to MHC-presentation of peptide antigens with subsequent activation of antigen-specific T-cell hybridomas. Taken together, these results demonstrate effective activation of the adaptive immune system in vitro. This synthetic platform can be used to build new vaccines for many different diseases for which immunodominant peptide antigens are known because the antigens can be quickly interchanged while the MS2 scaffold remains the same. Additionally, this platform allows for multiple peptide antigens to be delivered simultaneously in each capsid, which could provide enhanced immunity against resistant strains and be useful for cancer vaccine development.

biochemistry↗

bioRxiv2025

Conditional Protein Structure Generation with Protpardelle-1c

We present Protpardelle-1c, a collection of protein structure generative models with robust motif scaffolding and support for multi-chain complex generation under hotspot-conditioning. Enabling sidechain-conditioning to a backbone-only model increased Protpardelle-1cs MotifBench score from 4.97 to 28.16, outperforming RFdiffusions 21.27. The crop-conditional all-atom model achieved 208 unique solutions on the La-Proteina all-atom motif scaffolding benchmark, on par with La-Proteina while having ~10 times fewer parameters. At 22M parameters, Protpardelle-1c enables rapid sampling, taking 40 minutes to sample all 3000 MotifBench backbones on an NVIDIA A100-80GB, compared to 31 hours for RFdiffusion.

bioengineering↗

bioRxiv2025

Buyang Huanwu Decoction Ameliorates Peripheral Nerve Injury by Promoting Cell Cycle through Activation of PI3K/AKT/mTOR/HIF-1α Pathway

BackgroundPeripheral nerve injury (PNI) can lead to partial or even total motor and sensory dysfunction, severely affecting the work and life of patients, but recovery of nerve function is often poor due to the complexity of the nerve repair and regeneration process. Cumulative evidence suggests the contribution of cell cycle regulation in repairing PNI injury, and p27KIP1 is a key molecule in regulating the cell cycle. ObjectiveThe aim of this study is to elucidate the repair mechanism of peripheral nerve injury (PNI) from the point of view of regulating cell cycle and to explore the therapeutic effect of Buyang Huanwu Decoction (BHD). To provide new strategies for the treatment of PNI and to explore valuable drugs initially. MethodsThe clamp injury model was chose as a PNI model and different doses of BHD extract were administered by gavage for 14 days. Limb motor function, sciatic nerve index and sciatic nerve pathology were assessed and p27Kip expression level was determined. The in vivo chemical composition of BHD extract was determined and drug targets were retrieved by TCMSP, STITCH and Swiss Target Prediction to predict the mechanism of BHD treatment for PNI using network pharmacology. Pathway-related proteins including P-PI3K, P-AKT, P-mTOR and HIF-1a were validated by western blotting assay. ResultsCompared with control rats, PNI rats had abnormal motor function, sciatic nerve damage and reduced p27Kip expression. Administration of BHD for 14 days significantly improved PNI in rats, which not only repaired sciatic nerve function, attenuated sciatic nerve pathology and inhibited p27Kip expression to regulate the cell cycle in PNI rats, but also activated the PI3K/AKT/mTOR/HIF-1a pathway. ConclusionThe above results suggest that PI3K/AKT/mTOR/HIF-1a mediated cell cycle regulation may be an important repair mechanism for PNI, and that BHD can improve PNI by accelerating the cell cycle to promote neural repair and regeneration.

cell biology↗

bioRxiv2025

Structural Basis for Cas9-Directed Spacer Acquisition in Type II-A CRISPR-Cas Systems

CRISPR-Cas systems confer prokaryotic adaptive immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-EM structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo-state structure (Cas92-Csn28-Cas18-Cas24) adopts a resting conformation, with Cas9 locked in a nuclease-inactive state and Cas1-Cas2 sterically blocked from prespacer loading. Upon DNA engagement, Cas9 undergoes a conformational transition, forming a prespacer catching complex that threads the DNA through Csn2s central channel. This architecture enables Cas9 to interrogate the PAM sequence while sliding along the DNA, with Cas9 and Csn2 jointly define a 30-bp DNA segment which matches the prespacer length. Subsequent dissociation of Cas9 triggers a structural reconfiguration of the Csn2-Cas1-Cas2 assembly. The PAM-proximal DNA becomes accessible, and Cas1-Cas2 relocates to bind to the exposed DNA, enabling further prespacer processing and directional integration. These findings reveal how Cas9 collaborates with Csn2 and Cas1-Cas2 to couple PAM recognition with prespacer selection, resolving the dynamic structural transitions that ensure fidelity during type II-A CRISPR adaptation.

molecular biology↗

bioRxiv2025

HEAL-KGGen: A Hierarchical Multi-Agent LLM Framework with Knowledge Graph Enhancement for Genetic Biomarker-Based Medical Diagnosis

The discovery and validation of genetic biomarkers across diverse diseases demand intelligent systems capable of integrating complex multi-omics data with clinical relevance. We introduce HEAL-KGGen, an end-to-end framework that enhances Large Language Models (LLMs) through a hierarchical multi-agent architecture and an automatically constructed medical knowledge graph. The system includes a General Practitioner (GP) agent for initial biomarker triage and specialist agents for genomics, transcriptomics, proteomics, and clinical interpretation. The core innovation of HEAL-KGGen lies in its dynamic knowledge graph pipeline, which combines entity extraction based on patterns and semantics, ontology-aligned normalization (using UMLS, MeSH, SNOMED CT) and the construction of multi-source relationships from biomedical databases and literature. Retrieved subgraphs are transformed into contextual prompts that guide LLM reasoning via structured, explainable pathways. Our experiments show that HEAL-KGGen significantly improves question-answering accuracy across multiple mainstream large language models, with the highest improvement observed on Claude 3.5 Sonnetachieving a 43.75% increase in accuracy., confirming the value of domain-specific graph knowledge in advancing LLM performance for genetic and molecular diagnostics.

bioinformatics↗

bioRxiv2025

Stick-slip motion and universal statistics of cargo transport within living cells

Cargo transport within cells is a vital biological process that relies on the intricate interplay between motor proteins, microtubules, and the complex intracellular environment. In this study, we unveil a universal transport mechanism characterized by stick-slip motion, which governs the dynamics of intracellular vesicle transport. By analyzing a comprehensive dataset of vesicle trajectories across various cell types and intracellular environments, we demonstrate that the cargo velocities consistently follow a Gamma distribution, revealing a common statistical pattern amidst the diversity of biological cargoes. Our experimental findings are well-described by a theoretical model that connects the Brownian-correlated kinetic friction between motor-cargo complexes and their surroundings to the observed universal Gamma distribution of cargo velocities. This model elucidates the stick-slip dynamics governing intracellular cargo transport, which are pertinent to various cellular processes such as vesicle budding, organelle transport, and cell migration.

biophysics↗