Biology subjects

Lin, G.

Publications and source records attributed to Lin, G..

At least 19 recordsLinked to original sources

bioRxiv2026

A T2T Benchmark Reveals How Reference Choice Shapes Human Genome Interpretation

The completion of telomere-to-telomere (T2T) human genomes has expanded the accessible landscape of human genetic variation, yet benchmark resources remain limited to conventional high-confidence regions defined by existing reference frameworks. Here, we generated a near-perfect diploid T2T genome (T2T-LIN) from a Chinese individual and established assembly-based truth sets by comparison with T2T-YAO, an ancestry-matched near-perfect T2T reference genome. The benchmark showed a heterozygous/homozygous SNV ratio of ~2, consistent with expectations under Hardy-Weinberg equilibrium, and enabled genome-wide evaluation of reference-dependent biases. We found that reference choice substantially influences genome interpretation: ancestry-matched linear T2T references provided the most faithful representation of individual genomic variation and enabled more accurate genome reconstruction than unmatched linear, diploid and graph-based references. Benchmarking previously inaccessible repetitive and structurally complex regions revealed substantial limitations of current variant callers that were masked by conventional metrics. The T2T-LIN and YAO-LIN benchmarks establish a T2T-era framework for evaluating reference-dependent genome interpretation and variant discovery across nearly the complete human genome.

genomics↗

bioRxiv2026

Rapid Vascular Activation Precedes Immune Cell Infiltration Following Corneal Alkali Burn

Under homeostatic conditions, the cornea is avascular and contains few immune cells, but this changes rapidly following injury. Although the long-term consequences of corneal damage are well characterized, the earliest vascular and immune responses remain poorly understood. Here, we used a murine corneal alkali-burn model to examine limbal vascular activation and leukocyte recruitment immediately and at 2, 6, and 24 hours after injury. Limbal blood vessels underwent immediate dilation; however, vascular leakage into the corneal stroma occurred only in males. Lymphatic capillaries rapidly formed directed extensions toward the injury without significantly increasing their total vascular area, with males exhibiting longer extensions than females. Fluorescent dextran uptake provided evidence that these lymphatic vessels were functionally engaged in early tracer drainage. Despite pronounced vascular activation, early recruitment of neutrophils, monocytes, dendritic cells, macrophages, T cells, B cells, and natural killer cells remained limited. Thus, limbal blood and lymphatic vessels initiate the earliest response to corneal alkali injury before substantial leukocyte infiltration. These findings reveal sex-dependent differences in vascular permeability and lymphatic remodeling and identify the limbal vasculature as an early regulator of corneal inflammation and tissue repair.

pathology↗

bioRxiv2026

Non-Invasive Embryo Quality Assessment via Matrix-Optimized Untargeted LC-MS Metabolomics of Spent Embryo Culture Media and Weighted Ensemble Machine Learning

BackgroundNon-invasive embryo quality assessment is a critical unmet need in assisted reproductive technology (ART). Preimplantation genetic testing for aneuploidy (PGT-A) is effective but requires invasive biopsy that may compromise embryo viability. Metabolomics of spent embryo culture media (SECM) offers a non-invasive alternative, yet analytical challenges--limited sample volume, high salt content, and abundant proteins--have hindered standardization and clinical translation. ResultsWe systematically optimized sample preparation for untargeted LC-MS metabolomics of SECM using human serum as a reference. Optimal conditions were highly matrix-dependent: SECM required 7x volume of 50% acetonitrile for extraction and 40% acetonitrile for reconstitution, whereas serum required 10x volume of 100% methanol and 100% water--reflecting that SECM contains more non-polar species than serum. Applying the optimized workflow to 120 clinical SECM samples (72 euploid, 48 aneuploid), we identified 102 differential metabolites between euploid and aneuploid embryos, with prominent enrichment of lipid pathways (fatty acid metabolism, {beta}-oxidation, sphingolipid metabolism) and involvement of amino acid (methionine, tryptophan) and TCA cycle metabolism. A weighted ensemble machine learning model discriminated aneuploid from euploid embryos with an AUC of 0.977, 100.0% specificity, and 89.6% sensitivity. Among 72 euploid embryos stratified by morphological grading (good, fair, poor), metabolic alterations progressed from mitochondrial energy deficiency (good vs. fair) to broader lipid dysregulation (fair vs. poor), with the ensemble model achieving AUCs of 0.944, 0.889, and 0.943, respectively. ConclusionsThis study establishes a rigorously optimized and validated SECM metabolomics workflow that overcomes key analytical barriers in this challenging matrix. Our findings demonstrate that metabolic signatures--particularly in lipid and energy metabolism--are strongly associated with both embryo ploidy and morphological quality, providing biological insights into the metabolic underpinnings of embryo developmental competence. The high predictive performance of the ensemble model supports the feasibility of non-invasive embryo assessment as a complementary tool to existing methods, with potential to reduce reliance on invasive biopsy in ART. External validation in prospective multi-center cohorts is warranted to further assess clinical utility and generalizability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/741666v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@10fbe40org.highwire.dtl.DTLVardef@936273org.highwire.dtl.DTLVardef@246e5dorg.highwire.dtl.DTLVardef@1c9d115_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2026

UniFlow: Unifying protein conformational ensemble generation and machine-learned force fields with a scalable normalizing Flow

AO_SCPLOWBSTRACTC_SCPLOWMolecular dynamics (MD) provides a principled method for modeling equilibrium protein conformational energy landscapes, but its computational cost limits access to long timescales and larger protein systems. Recently, generative protein ensemble models and machine-learned coarse-grained force fields have emerged as complementary approaches for accelerating conformational sampling. However, they are typically developed separately despite modeling the same underlying equilibrium distribution. We introduce UniFlow, the first scalable generative model that unifies protein ensemble generation and machine-learned coarse-grained force fields for molecular dynamics simulation within a single framework. UniFlow employs an internal-coordinate normalizing flow that supports efficient i.i.d. sampling, exact likelihood evaluation, and differentiable energy and force computation. Across diverse protein systems, UniFlow generates ensembles that closely match reference MD simulations, generalizes to proteins beyond its training dataset, and samples substantially faster than diffusion-based ensemble-generation baselines. The same learned density further enables stable long-timescale molecular dynamics simulations. Together, UniFlow paves the way for a unified class of models that bridges generative ensemble modeling with physics-based molecular simulation. Codehttps://github.com/Harrydirk41/UniFlow.git

biochemistry↗

bioRxiv2026

Cerebrovascular Single-Nucleus RNA-Seq Reveals Heat Shock Activation and Vascular Remodeling in Alzheimer's Disease and Primary Tauopathies

Cerebrovascular alterations are widely observed in both Alzheimers Disease (AD) and primary tauopathies. Here, we hypothesized that mechanisms of cerebrovascular alterations are shared between AD and primary tauopathies. We performed single-nucleus RNA sequencing of postmortem human inferior temporal gyrus to characterize transcriptomic changes across cerebrovascular cell types in AD and primary tauopathies (Corticobasal Degeneration, Picks disease, and Progressive Supranuclear Palsy). Differential gene expression analyses revealed disease-specific transcriptional programs across vascular cell populations. However, genes involved in the heat-shock response were consistently upregulated across all diseases, suggesting a conserved cerebrovascular stress response during neurodegeneration. We further identified marked cerebrovascular remodeling in AD relative to primary tauopathies, along with dysregulation of genes mapping to AD risk loci in endothelial cells. Transcriptomic findings were validated using tissue clearing, light-sheet microscopy, and immunofluorescence quantification of vascular markers. These results define a conserved vascular stress program alongside AD-specific remodeling, highlighting the vasculature as a therapeutic target in neurodegeneration.

neuroscience↗

bioRxiv2026

ConformFlow: scalable normalizing flow for protein conformational ensemble generation

AO_SCPLOWBSTRACTC_SCPLOWMolecular dynamics (MD) simulations remain the standard tool for characterizing protein conformational landscapes, but their high computational cost limits large-scale and long-timescale applications. Recent generative models, especially diffusion-based approaches, provide promising alternatives by learning equilibrium conformational distributions across diverse protein systems. We present ConformFlow, the first scalable normalizing-flow framework for sequence-conditioned protein conformational ensemble generation. ConformFlow combines a continuous backbone latent representation with a RealNVP-style flow parameterized by sequence-aware Transformer coupling networks, enabling exact likelihood training, single-step sampling, and plug-and-play conditioning on flexible geometric constraints. Across diverse protein systems, ConformFlow generates ensembles that agree well with reference MD simulations, generalizes to proteins beyond its training data, and achieves substantially faster sampling than diffusion-based baselines. These results establish ConformFlow as an efficient and controllable alternative for protein conformational ensemble generation. Codehttps://github.com/Harrydirk41/ConformFlow.git

biochemistry↗

bioRxiv2026

RAI1 safeguards fidelity and tempo of human neurodevelopmental gene expression

Human brain development proceeds on an unusually long timeline relative to other species, a feature that is thought to foster advanced cognitive abilities. The Retinoic Acid Induced 1 (RAI1) gene encodes a nucleosome-binding protein, and its haploinsufficiency is responsible for Smith-Magenis Syndrome (SMS), a neurodevelopmental disorder characterized by cognitive impairment with autistic features. However, the role of RAI1 in human neurodevelopment remains unexplored experimentally. Here, we generated isogenic heterozygous and homozygous RAI1 loss-of-function human embryonic stem cell lines and interrogated the roles of RAI1 in neurodevelopmental gene regulation. A longitudinal transcriptome analysis during in vitro cortical development revealed that RAI1 deficiency accelerates the progression of developmental gene expression. Single-cell RNA-seq analysis revealed that RAI1-deficient neuroprogenitors acquire a transient mesoderm-like gene expression signature, followed by a pro-neuronal maturation signature in postmitotic neurons. Unexpectedly, the developmental acceleration signature was exacerbated during NGN2-induced excitatory neuron differentiation, isolating the roles of RAI1 in neuronal differentiation from non-neuronal functions. Together, these results identify RAI1 as a suppressor of the mesodermal lineage program and as a brake that slows the tempo of human neurodevelopmental gene expression.

neuroscience↗

bioRxiv2026

Evolution of supernumerary chromosomes in wheat blast fungal pathogens

A genome of Pyricularia oryzae (synonym Magnaporthe oryzae), the fungus that causes blast disease on diverse grass species, has seven core chromosomes and may contain supernumerary mini-chromosomes. The P. oryzae Triticum (PoT) pathotype is the phylogenetic lineage responsible for devastating epidemics of wheat blast disease. Genomic analysis of wheat blast field isolates from the initial outbreak in 1985 in Brazil through recent field isolates in South America revealed dynamic presence and structure of mini-chromosomes. Two "earliest" field isolates representing founder lineages for the Triticum pathotype contain similar mini-chromosomes. Another PoT founder isolate from 1986 and 37 out of 39 Triticum field isolates collected between 1986 and 1992 lack mini-chromosomes. Mini-chromosomes present in the founder strains each contain two copies of the PWT7 wheat blast avirulence gene, and PWT7 was lost from subsequent early strains through mini-chromosome loss. Almost all PoT field isolates from 2005 to 2020 have regained mini-chromosomes in which PWT7 sequences have been replaced by other sequences. Telomere-to-telomere assemblies of 11 mini-chromosomes identified two major mini-chromosome types in the South American PoT population, and demonstrated significant within-mini-chromosome sequence alterations as well as recombination with other mini-chromosomes or core chromosome ends. Additionally, our data indicate horizontal mini-chromosome transfer between Pyricularia species, resulting in nearly identical genomic fragments shared between P. oryzae and Pyricularia pennisetigena isolates in the PWT4 avirulence gene region. Our genomic analysis depicts the dynamic mini-chromosome compartment in the diverse South American Triticum field population through time, indicating important roles for mini-chromosomes in pathogen adaptation and pathogenicity.

evolutionary biology↗

bioRxiv2026

Unlocking Ultrasensitive Phosphoproteomics of Nanoscale Samples: A Rapid IMAC Enrichment Platform Using Ti-PAN Tip

Protein phosphorylation is a critical mechanism in cellular signal transduction, playing essential roles in various cellular processes. However, significant challenges of their low abundance, high dynamic range, and complexity are especially prominent for accurate phosphoproteome analysis in nanoscale samples, such as oocytes, where misregulation of phosphorylation can lead to infertility. In this study, we developed a novel phosphopeptide enrichment tip (Ti-PAN) using highly hydrophilic textile PAN fibers, enabling rapid in-situ one-tip IMAC enrichment in just 8 minutes. This approach not only minimizes sample loss but also enhances analytical sensitivity and is easily adaptable to automation for high-throughput applications, demonstrating excellent recovery (>94%) and outstanding selectivity for phosphopeptides with remarkable resistance to interference up to 1:5000. By integrating it with a compatible sample preparation workflow based on n-dodecyl {beta}-D-maltoside (DDM) lysis with the high-sensitivity MS data acquisition method of turboDDA, comprehensive mapping of phosphosites in microsamples was achieved, including those from the ERK interactome, mouse brain slices, and oocytes. Using this approach, we identified 2,139 to 11,296 phosphosites across 645 to 2,168 phosphoproteins from samples ranging from 100 to 100,000 cells. Furthermore, we successfully applied this strategy to analyze the phosphoproteome of mouse oocytes at different developmental stages, using as few as 5 oocytes per sample. This led to the identification of 2,709 phosphosites on 701 phosphoproteins, revealing several key kinases involved in meiotic resumption and oocyte maturation, including STK4 (serine/threonine kinase 4) and DYRK3 (dual specificity tyrosine phosphorylation-regulated kinase 3). To our knowledge, this represents the deepest coverage of phosphoproteomes from such minimal oocyte material reported to date, offering valuable mechanistic insights into developmental controls during oocyte maturation. This Ti-PAN-based method provides a robust, versatile, and automatable platform for large-scale, highly sensitive phosphopeptide enrichment, substantially advancing phosphoproteomic studies in complex biological systems.

biochemistry↗

bioRxiv2026

All-in-one Single-Cell Proteomic Analysis of Protein Alterations in Human Oocytes Undergoing in Vitro Aging

Study questionWhat specific proteomic changes and molecular markers associated with in vitro aging occur in human oocytes? Summary answerSingle-cell proteomic analysis revealed the protein dynamics during the in vitro aging of oocytes. Subsequent functional analysis highlighted key biological processes affected in aged oocytes and identified several genes as candidate key factors for oocyte and early embryo quality. What is known alreadyOvulated oocytes undergo a time-dependent degradation process if fertilization does not occur within a specific window. This aging process leads to morphological, molecular, and epigenetic changes that can compromise oocyte quality and subsequent embryo development. In previous studies, transcriptome sequencing in humans and mice has revealed serial changes in aging oocytes including oxidative stress, mitochondrial dysfunction, DNA damage, and alterations in cell cycle regulation. These changes can lead to apoptosis, chromosomal abnormalities, and epigenetic modifications, all of which negatively affect oocyte quality. However, research regarding protein level changes and regulatory mechanisms in human oocytes during in vitro aging remains unclear. Study design, size, durationUtilizing "All-in-one" single-cell proteomics, we investigated the protein expression levels in human oocytes at the germinal vesicle (GV) stage and metaphase II (M II) stage following 24 hours of in-vitro aging. We analyzed four groups of oocytes: fresh GV (n=8), in vitro-aged GV (n=9), fresh M II (n=11), and in vitro-aged M II (n=11). This approach allowed for a detailed comparison of proteomic changes associated with in vitro aging in oocytes at different developmental stages. Participants/materials, setting, methodsOocytes at GV and MII stage were collected for "All-in-one" single-cell quantitative proteomic respectively. Differentially expressed genes were analyzed using the R package DESeq2 or DEP (filtered with q-value[≤]0.05 and Foldchange[≥]1.5). EGSEA package was used to perform pathway analysis. Main results and the role of chanceThe proteomic analysis of fresh and in vitro-aged human GV and MII oocytes identified 3,268 proteins. In GV oocytes, compared to fresh samples, 73 proteins were upregulated and 90 were downregulated. Gene Ontology (GO) enrichment analyses revealed these DEPs were involved in key biological processes such as lysosome organization, spindle assembly and oxidative stress response. Gene Set Enrichment Analysis (GSEA) revealed a significant down regulation of genes associated with protein translation initiation and methylation pathways, while those in the apoptosis mitochondrial changes were upregulated. In MII oocytes, 63 proteins were upregulated and 69 were downregulated. GO analysis highlighted their involvement in cytoplasmic translation, ribosome biogenesis and oocyte development. GSEA showed that aged MII oocytes exhibited a downregulation of gene sets in protein translation but an upregulation in membrane fusion pathways compared to fresh oocytes. Furthermore, we identified proteins with consistent expression trends across both GV and MII stages, including MRFAP1 and MT2A, as candidate biomarkers for human oocyte aging in vitro. Limitations, reasons for cautionSingle cell proteomic techniques may not fully capture the dynamic range of proteins present within oocytes, leading to potential underrepresentation of low-abundance proteins that could play crucial roles. Furthermore, the heterogeneity within oocytes introduces variability that can obscure the identification of consistent proteomic signatures associated with oocyte quality or aging. Wider implications of the findingsThese findings not only provide an understanding of the protein changes underlying the in vitro aging of human oocytes but also offer potential biomarkers and intervention targets for future research and clinical applications in assisted reproductive technologies. Study funding/Competing interest(s)This project received funding from the National Natural Science Foundation of China (NO.82471693, 22574175); the Natural Science Foundation of Hunan Province (NO. 2024JJ4100), the Hunan Provincial Grant for Innovative Province Construction (2019SK4012), the Hundred Youth Talents Program of Hunan Province (to S.Z.), the Major Scientific Program of CITIC Group (No. 2023ZXKYB34100) and Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-XIANGYA (YNXM-202313, YNXM-202319, YNXM-202211). The authors have no conflicts of interest to declare.

developmental biology↗

bioRxiv2026

Rapid sensing and relaying of cellular hyperosmotic-stress signals via RAF-SnRK2 core condensates

Hyperosmolarity caused by drought, high salinity, or cold stress inhibits plant growth and crop productivity1,2. A conserved protein-kinase cascade of cytosolic B-RAFs and SnRK2s is rapidly activated upon osmotic stresses to initiate downstream adaptive responses, which represents one of the fastest known responses to osmotic stress in plants3-8. How the kinase cascade is activated by osmotic stress is unknown. Here, we show that Arabidopsis B4 subgroup RAFs have intrinsically disordered regions and directly sense both ionic and non-ionic hyperosmolarity by reversible condensation. B4-RAFs recruit and co-condense with subclass-I SnRK2s to phosphorylate and turn on SnRK2s, evading the non-condensable inhibitory A-clade PP2C phosphatases. This straightforward osmosensing and relaying module can be fully reconstituted in E. coli by co-expressing three components or in solution in a test tube using recombinant proteins. Our findings identify B-RAFs as the chief cellular osmosensors that detect low water potential by co-condensation, forming a signal hub with SnRK2s to orchestrate adaptive responses in plants, and represent an evolutionarily conserved osmosensing mechanism across kingdoms.

plant biology↗

bioRxiv2025

Stage-specific regulation of the Plasmodium falciparum proteasome activity reveals adaptive rewiring in artemisinin resistance

The ubiquitin-proteasome system (UPS) is essential for Plasmodium falciparum to maintain protein homeostasis, adapt to proteotoxic stress, and regulate parasite growth and stage transitions. The proteolytic 20S proteasome core is the central component of the UPS, where unfolded protein substrates are degraded into oligopeptides. Mechanisms regulating malaria parasite proteasome activity are poorly understood and have not been thoroughly studied. This knowledge gap is especially critical in the context of artemisinin (ART) resistance, where parasite survival depends on an enhanced stress response, including a greater reliance on the UPS. Here, we profiled proteasome activity and abundance across the parasite intraerythrocytic developmental cycle (IDC) in both ART-sensitive (ART-S) Dd2 and ART-resistant (ART-R) Dd2K13R539T parasites. We uncovered striking stage-specific regulation: proteasome activity was abundant in the ring stage, decreased in trophozoites, and then peaked in schizonts. Furthermore, ART-R Dd2K13R539T parasites exhibited higher ring-stage proteasome activity than ART-S Dd2, despite reduced proteasome abundance, suggesting a unique adaptive rewiring of proteasome function. To study proteasome regulation in the parasite, we manipulated proteasome abundance in Dd2 and Dd2K13R539T by creating a conditional knockdown of PfUMP1, a conserved proteasome maturation factor. PfUMP1 depletion disrupted 20S assembly, decreased proteasome activity, and led to parasite death. These experiments uncovered two key features of proteasome regulation in P. falciparum: (1) the absence of canonical transcriptional regulation of proteasome genes in response to downregulation of proteasome activity, and (2) ART-R parasites exhibit a ring-stage specific increased sensitivity to proteasome downregulation. Together, our findings reveal a previously unrecognized layer of proteasome regulation in malaria parasites and how, as part of their survival adaptations to decreased hemoglobin uptake associated with ART resistance, these parasites alter proteasome function to survive. This work reinforces the therapeutic potential of the proteasome as a stage- and resistance-specific antimalarial target.

microbiology↗

bioRxiv2025

Complete and haplotype-resolved maps of genomic and epigenetic discordance in monozygotic twins

Telomere-to-telomere (T2T) genome assemblies are indispensable for accurate detection of genetic variation and for resolving complex repetitive regions. Monozygotic (MZ) twin pedigrees provide a powerful model to investigate de novo mutations (DNMs), however, comprehensive, haplotype-resolved analyses of structural variation (SV), allele-specific inheritance in complex regions, and DNA methylation in diploid human genomes remain limited. Here, we generated complete, haplotype-resolved T2T assemblies for two female twins (C33 and C35) from a Han Chinese pedigree by integrating complementary, state-of-the-art sequencing technologies. The resulting T2T-C33 and T2T-C35 assemblies are highly contiguous and complete, with Genome Continuity Inspector (GCI) scores of 74.94 (maternal) and 77.94 (paternal), and consensus quality values (QV) >75 (k = 21). We comprehensively cataloged 62 inter-twin single-nucleotide variants (SNVs), 15 small indels, and identified both shared and private DNMs, revealing nascent genomic divergence between the MZ twins. Focused interrogation of complex regions uncovered pronounced haplotype-specific length polymorphisms and structural heterogeneity within centromeric higher-order repeat (HOR) arrays. Notably, we observed extensive HOR copy-number variation between haplotypes, including a large copy-number difference on maternal chromosome 18, underscoring dynamic HOR array evolution even among genetically identical individuals. Concurrently, genome-wide DNA methylation profiling delineated allele-specific epigenetic variation that may contribute to phenotypic discordance. Together, these high-quality, diploid T2T genomes from a Han Chinese pedigree provide a valuable resource for population-aware genomics and reveal fine-scale, haplotype-specific divergence in MZ twins. Our results advance understanding of repeat dynamics, centromeric architecture, epigenetic variation and the spectrum of human genomic variation at single-base and structural scales.

genomics↗

bioRxiv2025

CDK4/6 Inhibition Reverses MEIS2 Suppression of CRL4 to Enhance Immunomodulatory Drug Therapy in Multiple Myeloma

MEIS2 was identified biochemically as a substrate of cereblon (CRBN), a receptor of the CRL4CRBN E3 ubiquitin ligase required for the anti-myeloma activity of immunomodulatory drugs (IMiD)s and CELMoDs. However, its function in myeloma is unknown. We discovered that MEIS2 is aberrantly expressed in bone marrow myeloma cells (BMMC)s, and that high MEIS2, CDK4 or CDK6 and low CRBN expression predisposes patients to inferior overall survival in IMiD therapy. Inhibition of CDK4/6 (CDK4/6i) reprograms BMMCs for IMiD vulnerability ex vivo that mimics patients response to IMiDs. Mechanistically, CDK4/6i both rapidly accelerates the displacement of MEIS2 from CRBN by IMiD and destabilizes the MEIS2 protein while increasing the CRBN protein in cooperation with IMiD and CELMoD. This enhances CRL4CRBN ubiquitination of IKZF3 and IKZF1 for degradation that exacerbates the loss of IRF4 to relieve IRF7 for induction of the interferon response, culminating in TRAIL-mediated apoptosis. Additionally, MEIS2 promotes BCMA expression and antagonizes repression of BCMA by IMiD and CELMoD for survival of myeloma cells. Thus, CDK4/6i reverses MEIS2 inhibition of CRL4CRBN in cooperation with IMiD and CELMoD, and mitigates MEIS2-mediated BCMA signaling for survival, suggesting targeting MEIS2 and CDK4/6 as a new strategy to advance immunomodulatory drug therapy in multiple myeloma.

cancer biology↗

bioRxiv2025

ProTDyn: a foundation Protein language model for Thermodynamics and Dynamics generation

AO_SCPLOWBSTRACTC_SCPLOWMolecular dynamics (MD) simulation has long been the principal computational tool for exploring protein conformational landscapes and dynamics, but its application is limited by high computational cost. We present ProTDyn, a foundation protein language model that unifies conformational ensemble generation and multi-timescale dynamics modeling within a single framework. Unlike prior approaches that treat these tasks separately, ProTDyn allows flexible independent and identically distributed (i.i.d.) ensemble sampling and dynamic trajectory simulation. Across diverse protein systems, ProTDyn yields thermodynamically consistent ensembles, faithfully reproduces dynamical properties over multiple timescales, and generalizes to proteins beyond its training data. It offers a scalable and efficient alternative to conventional MD simulations. Code is available at: https://github.com/Harrydirk41/ProTDyn.

biochemistry↗

bioRxiv2025

Time-Resolved Crystallography Reveals the Mechanisms of GTP hydrolysis for N-RAS and the Oncogenic Mutants G12C, G12V and Q61L

The RAS family of small GTPases are molecular switches that convey downstream signals regulating cell proliferation, differentiation, and apoptosis. The signaling competent GTP-bound RAS transitions to its inactive GDP-bound form through {gamma}-phosphate hydrolysis. Oncogenic RAS mutations hamper GTP hydrolysis and are present in up to 30% of all human cancers. Structural studies of RAS proteins bound to non-hydrolysable GTP analogs have revealed snapshots of the enzyme in its possibly active form. Yet, the mechanism of GTP hydrolysis has not been structurally resolved. To visualize this reaction in real time, we performed time-resolved crystallographic experiments employing a photolabile caged-GTP substrate. Fifty-seven distinctive reaction intermediates were captured during hydrolysis of a live GTP for N-RAS, the oncogenic mutants G12C, G12V and Q61L; and Y32R, a fast hydrolytic mutant. The reaction mechanisms and rates for the native and each of the mutants differed significantly; however, they shared common elements: an initially catalytically-defective open state, which transitions into the closed Michaelis complex state with solvent-assisted O3B-P{gamma} bond lengthening and breaking, followed by the release of the Mg2+ stabilized PO3-/PO4-3 species and unfolding of the switch loops. Given the conserved nature of GTP- and ATP-ases active sites, this structural work lays the basis to understand the universal mechanism of {gamma}-phosphate hydrolysis. Furthermore, search for cryptic binding sites during GTP hydrolysis in G12C, G12V, and Q61L mutants reveals the presence of distinctive state-dependent binding pockets that could be targets for structure-based drug discovery of experimentally resolved intermediates states.

cancer biology↗

bioRxiv2025

Recombinant proteasome provides new avenues for anti-malarial drug development.

The Plasmodium falciparum 20S proteasome (Pf20S) has emerged as a promising antimalarial target. Development of therapeutics to this target has previously relied on native purifications of Pf20S, which is challenging and has limited the scope of previous efforts. Here, we report an effective recombinant Pf20S platform to facilitate drug discovery. Proteasome assembly was carried out in insect cells by co-expressing all fourteen subunits along with the essential chaperone homolog, Ump1. Unexpectedly, the isolated proteins consisted of both a mature and an immature complex. Cryo-EM analysis of the immature complexes revealed structural insights detailing how Ump1 and the propeptides of the {beta}2 and {beta}5 subunits coordinate {beta}-ring assembly, which differ from human and yeast homologs. Biochemical validation confirmed that {beta}1, {beta}2, and {beta}5 subunits of the mature proteasome were catalytically active. Clinical proteasome inhibitors, bortezomib, carfilzomib and marizomib were potent but lacked Pf20S selectivity. However, the tripeptide-epoxyketone J-80 inhibited Pf20S {beta}5 with an IC50 of 22.4 nM and 90-fold selectivity over human {beta}5. Structural studies using cryo-EM elucidated the basis for the selective binding of J-80. Further evaluation of novel Pf20S-selective inhibitors such as the reversible TDI-8304 and irreversible analogs, 8304-vinyl sulfone and 8304-epoxyketone, confirmed their potency and selectivity over the human constitutive proteasome. This recombinant Pf20S platform facilitates detailed biochemical and structural studies, accelerating the development of selective antimalarial therapeutics.

biochemistry↗

bioRxiv2025

Group size and social interactions' strength determine collective response to perturbation in a model of burst-and-coast swimming fish

Collective responses to localized perturbations are essential for the adaptability of animal groups. Using a biologically grounded computational model of burst-and-coast swimming in Hemigrammus rhodostomus, we investigate how group size and the strength of social interactions shape collective dynamics under perturbation. The model integrates experimentally derived attraction and alignment rules, behavioral heterogeneity, and boundary effects within a circular tank. We identify four collective states (schooling, milling, turning, and swarming) and characterize a critical regime in which groups exhibit multistable dynamics. At this critical point, small subsets of perturbing individuals, defined by altered social interaction strengths, can induce sharp transitions to new collective states. In particular, such transitions occur in large groups (N = 100) but not in smaller ones (N = 25 or 50), highlighting a size-dependent sensitivity to disturbance. We show that both the nature of the perturbing individuals and the initial state of the group modulate the systems responsiveness. Our findings suggest that large groups may exploit criticality to remain both robust, flexible, and that individual variability can serve as a catalyst for adaptive reconfiguration. This work provides new insights into how the internal group structure and perturbation design influence collective behavior in animal groups.

animal behavior and cognition↗