Biology subjects

Zhang, T.

Publications and source records attributed to Zhang, T..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

Targeting homologous recombination repair to potentiate fluoroquinolone efficacy in Mycobacterium abscessus

Synthetic antibiotics, fluoroquinolones (FQs), are extensively important treatment options for fast growing mycobacterial infections. However, their widespread use has led to decreased efficacy due to intrinsic or acquired resistance. FQs exhibit antimicrobial activity by stabilizing type II topoisomerases-generated DNA breaks, thereby inducing lethal double-strand breaks (DSBs) and leading to bacterial death. Escalating resistance among mycobacterial pathogens highlights the need to elucidate resistance mechanisms and identify novel strategies to restore or enhance the drug activity. Here, we revealed homologous recombination (HR) as a key determinant of FQ tolerance in Mycobacterium abscessus, a notoriously drug-resistant pathogen. Through a transposon mutagenesis screen, we identified that disruption of adnB - an HR-associated gene, markedly sensitized M. abscessus to FQs. This finding prompted a systematic dissection of DSB repair pathways in M. abscessus. Targeted deletion of HR core components (adnB, recO, recA, recR, ruvB) significantly increased FQ susceptibility in vitro, while inactivation of alternative DSB repair pathways - single-strand annealing (SSA) and non-homologous end joining (NHEJ) - had no effect, revealing a unique reliance on HR for FQ tolerance. Furthermore, the complementation of gene knock-out M. abscessus strains {Delta}adnB, {Delta}recO, {Delta}recA, {Delta}recR or {Delta}ruvB with the corresponding genes from M. abscessus, as well as homologous genes from Mycobacterium tuberculosis restored the resistance phenotype, indicating a conserved HR-dependent tolerance mechanism in M. abscessuss. In a murine infection model, genetic abrogation of HR significantly improved the therapeutic efficacy of FQs against M. abscessus, demonstrating translational relevance. Collectively, our findings position HR as a conserved and actionable vulnerability in M. abscessus, and provide a compelling rationale for developing HR-targeted adjuvant therapies to resensitize drug-refractory strains to FQ treatment. Impact statementFluoroquinolone (FQ) resistance poses a major challenge in the treatment of Mycobacterium abscessus infections. This study identifies homologous recombination (HR) as a key defense mechanism that limits FQ efficacy across multiple mycobacterial species. Genetic inhibition of HR not only sensitizes M. abscessus to FQs in vitro but also enhances antibiotic activity in vivo. These results highlight HR repair as a promising therapeutic target to potentiate FQ activity and combat drug resistance in M. abscessus infections.

molecular biology↗

bioRxiv2025

Condensate-Driven Triglyceride Depletion Links α-Synuclein to Mitochondrial Dysfunction

Inclusions of -Synuclein (Syn) characterize multiple age-related neurodegenerative diseases, including Parkinsons disease (PD) and Multiple System Atrophy (MSA). While interactions between Syn and lipids are known to contribute to Syn pathobiology, the precise cellular mechanisms that link lipids to Syn toxicity have yet to be elucidated. Through lipidomic profiling of Caenorhabditis elegans, we found that Syn progressively alters lipid metabolism in aging worms. Syn strongly reduces overall content of triacylglycerols (TAG) and disrupts the structure of lipid droplets (LD). These pathological changes depend on Syns properties to condensate and form inclusions. Apart from lowering TAG levels, Syn also increases the proportion of long-chain unsaturated fatty acids (LCUFAs). Consequently, genetic inhibition of LCUFA biosynthesis alleviates Syn-induced loss of C. elegans motility. Strikingly, bypassing lipid metabolic defects by supplementing Medium Chain Fatty Acids (MCFAs) restores the Syn-impaired mitochondrial response and rescues motility. These results link Syn condensation to impaired TAG metabolism, which reduces mitochondrial function and enhances overall toxicity. Together with the finding that plasma TAGs are lowered in Parkinson patient cohorts, these results suggest that restoring TAG metabolism could alleviate Syn-induced toxicity in Parkinsons and other age-related synucleinopathies.

cell biology↗

bioRxiv2025

Sulcal landmarks reveal lineage-specific trajectories and multi-scale specialization of the mammalian cortex

An important topic of evolutionary neuroscience is to understand how brain function and structure across evolutionary time. In the absence of functional data for ancestral species, studies often rely on structural features such as cortical volume or surface area. However, atlas-based metrics derived from human or a few model organisms lack biological interpretability across species with divergent cortical architectures. Here, we investigate sulcal pits--the locally deepest points of cortical folds--as conserved, atlas-independent landmarks that provide a robust basis for cross-species comparison. Using cortical surface reconstructions from 90 mammalian species, we show that sulcal pit distribution patterns partly recapitulate phylogenetic relationships, independent of overall brain size (volume and surface area), and vary systematically with ecology and lifestyle. By tracing pit-based evolutionary trajectories from 80 million years ago to the present, we found pronounced differences across ecological and behavioral categories, indicating that cortical folding has diversified in close association with species habitats, lifestyles, and social structures. Using sulcal pits, we identified Homo sapiens-specific regions and species-shared regions. The Homo-specific areas were functionally associated with higher cognitive and emotional processes, distinguished by unique histological features, enriched for gene sets related to neural regulation, and exhibited markedly different cell-type abundance profiles compared to shared regions. Together, these results establish sulcal pits as a robust, evolutionarily informative feature for cross-species cortical alignment, offering new insights into the structural innovations underlying brain evolution study.

evolutionary biology↗

bioRxiv2025

Noncanonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy

We report a minimally disruptive labeling strategy for stress granule protein, G3BP Stress Granule Assembly Factor 1 (G3BP1), and ALS-linked protein, TAR DNA-binding protein 43 (TDP-43), using the fluorescent noncanonical amino acid Anap. By integrating genetic code expansion (GCE) with rational site selection, we achieved precise incorporation of Anap that preserves protein structure and function. In live cells and neurons, Anap labeling faithfully recapitulated localization, stress-induced dynamics, and recovery behavior, outperforming conventional fluorescent tags and enabling physiologically relevant visualization of protein pathobiology.

cell biology↗

bioRxiv2025

CTDP: Identifying cell types associated with disease phenotypes using scRNA-seq data

Single-cell RNA sequencing enables transcriptome-wide analysis at single-cell resolution, offering unprecedented insights into cellular heterogeneity across biological conditions. However, accurately comparing transcriptomic distributions of cells from distinct biological states, such as healthy versus diseased individuals, remains challenging. To address this, we developed CTDP, a robust and interpretable computational framework that identifies disease phenotype-associated cell types of interest by integrating Lasso-regularized logistic regression with permutation testing. Through comprehensive evaluations on both simulated and real-world datasets, including melanoma immunotherapy, COVID-19 severity, and liver cirrhosis, CTDP consistently outperformed existing methods such as DA-seq, scDist, and PENCIL in both accuracy and robustness. In melanoma, CTDP uncovered immune-responsive clusters and revealed transcriptional regulators like PTPRC, CREM, and JUNB linked to immunotherapy efficacy. In COVID-19, it identified critical severity-associated cell types, such as B cells, NK cells, epithelial cells, and macrophages, which contribute to dysregulated immune responses and inflammation in severe cases. These results highlight CTDPs power in uncovering disease-relevant cell populations and its potential to advance precision medicine through single-cell analysis.

bioinformatics↗

bioRxiv2025

AniMarkerDB: a comprehensive database for exploring cell types and marker genes in livestock and poultry at single-cell resolution

Single-cell RNA sequencing (scRNA-seq) has dramatically advanced the understanding of cellular heterogeneity. While numerous marker gene databases are available for humans and mice, a lack of systematic resources for livestock and poultry species remains, limiting progress in functional genomics, immunology, and breeding.. To address this challenge, we developed AniMarkerDB (https://animarkerdb.bio), a comprehensive and curated database dedicated to marker genes and immune-related epitopes in economically animals, including chicken, pig, and duck. AniMarkerDB integrates 7,010 marker gene across 37 tissues and 846 cell types, together with 71,442 immune epitope records from IEDB. All entries undergo rigorous literature curation, manual validation, and multi-level quality control, with standardized nomenclature and annotation to ensure data consistency and reusability. The platform supports flexible queries by species, tissue, cell type, or gene. It offers analytical tools for cross-species comparison model organisms such as human and mouse, interactive single-cell atlas visualization, and user-defined cell type annotation. Additionally, AniMarkerDB provides dynamic visualizations and export options, enabling researchers to efficiently obtain large-scale marker and epitope data for downstream applications such as infectious disease research, vaccine target design, and comparative immunology. Looking ahead, AniMarkerDB will expand species coverage and incorporate additional modalities, including single-cell atlases from healthy and disease models, establishing itself as a comprehensive and authoritative platform for animal cell biology, disease modeling, and translational research. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/682327v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@af7e7borg.highwire.dtl.DTLVardef@198d114org.highwire.dtl.DTLVardef@1c69ed2org.highwire.dtl.DTLVardef@e4fc43_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Effective repurposed antivirals against an emerging virus

The ongoing global monkeypox virus (MPXV) outbreak urgently needs effective medications, which be accelerated through drug repurposing. However, it is challenging to poinpoint protein targets. Here we introduce a novel method rooted in molecular evolutionary theory for quick drug target identification for MPXV. It identifies drug targets as positively selected genes of viral proteins which bind host proteins. From the identified targets, we select a top gene product OPG021 for virtural drug screening. One top-ranked drug nilotinib is experimentally shown to have a significant 69% of antiviral efficacy of the FDA-approved antiviral tecovirimat (TPOXX(R) or ST-246). Higher binding affinity but not antiviral efficacy of repursposed drugs than FDA-approved drugs suggests the complexity of drug repurposing and underscores the importance of experimental validation. This innovative drug target identification strategy will contribute to combating the ongoing MPXV outbreak and other viral acute and chronic viral diseases.

pathology↗

bioRxiv2025

Mechanisms of sex differences in acute and long COVID sequelae in mice

Males are more likely to suffer severe outcomes during acute COVID-19 and a greater proportion of females develop post-acute sequalae of COVID-19 (PASC). To identify mechanisms of PASC, mice were infected with SARS-CoV-2 and viral, inflammatory, and neurocognitive outcomes were evaluated through 84 days post-infection. Sex differences were not observed in virus replication or persistence of viral RNA in pulmonary or extrapulmonary tissues. After infectious virus clearance, females exhibited persistent neurocognitive impairments, and greater frequencies of inflammatory myeloid cells, neuroinflammation, and dysregulated T cells, including effector memory and Tregs. Sex differences in persistent inflammation and cognitive impairments during PASC were mediated by the presence of two X chromosomes and infection-induced upregulation of the X-linked genes Xist and Tlr7, which was reversed by pharmacological inhibition of TLR7 activity during the PASC phase. Sustained X-linked TLR7-mediated inflammation underlies how the burden of PASC is greater for females and provides a therapeutic target.

immunology↗

bioRxiv2025

Mapping the Telomeric 3D Interactome with Telomere-C Reveals Repetitive Element Hubs Associated with Telomere Maintenance

Telomeres are essential for genome integrity, but the accurate, high-resolution mapping of their three-dimensional (3D) chromatin interactions, a process thought to mediate gene regulation and telomere maintenance, has been limited by the repetitive nature of subtelomeric DNA and shortcomings of traditional sequencing-based chromatin conformation capture methodologies. To overcome these challenges, we developed Telomere Conformation Capture by sequencing (Telomere-C), a novel high-throughput approach for the genome-wide identification of telomeric chromatin interactions. We applied Telomere-C to three normal fibroblast cell lines (BJ, IMR90, WI38), one telomerase-positive cancer cell line (HeLa S3) and two cancer cell lines (U2-OS and WI38-VA13) that maintain telomeres by alternative lengthening of telomeres (ALT). We generated millions of telomere-associated reads per sample and identified up to 41,026 interaction peaks. We successfully validated previously-described telomeric interaction sites (e.g., TERT and DUX4), and critically, revealed a novel class of ultra-long-range interactions extending over 5 Mb away from the telomere, which surprisingly constitute over 80% of all identified contacts. Contrary to prior studies focusing on protein coding gene interactions, we discovered that the telomeric 3D interactome is overwhelmingly anchored at repetitive element hubs, particularly interstitial telomeric sequence (ITS), telomere-associated repeat 1 (TAR1) and D20S16 elements. We found that the clustering of these interactions correlates strongly with cell-type-specific telomere maintenance. Most notably, D20S16 telomeric interactions were uniquely and highly enriched in ALT cancer cells, suggesting a mechanistic link. Taken together, our study effectively constructs the first high resolution maps of the telomeric 3D interactome, redefining its scope to be dominated by ultra-long-range contacts with repetitive elements. This work provides fundamental insights into telomeric nuclear organization and establishes the telomeric D20S16 interaction as a molecular signature for the ALT pathway.

genomics↗

bioRxiv2025

Network pharmacology, bioinformatics, molecular docking and experimental verification of the mechanism of HXZTS treatment on osteoarthritis synovium

BackgroundOsteoarthritis (OA) is a prevalent joint disease characterized by synovial inflammation and synovial fibroblast dysfunction. Elucidating the molecular mechanisms underlying synovial tissue impairment in OA is crucial for developing targeted therapeutic interventions. Huoxuezhitongsan (HXZTS), a traditional Chinese medicine formula, has shown promising therapeutic effects in treating OA, but its mechanisms of action on synovial tissue remain unclear. MethodThis study integrated network pharmacology, bioinformatics analysis, molecular docking, and experimental validation to elucidate the potential targets, pathways, and mechanisms by which HXZTS exert therapeutic effects on osteoarthritic synovial tissue. ResultsNetwork analysis identified 21 active ingredients and 111 common targets of HXZTS for OA, with key compounds like quercetin, {beta}-sitosterol, and stigmasterol. Enrichment analysis revealed HXZTS modulates inflammation through TNF, IL-17, and MAPK signaling pathways, oxidative stress, apoptosis, angiogenesis, and metabolic regulation. Molecular docking demonstrated strong binding affinities of HXZTS compounds with targets like AKT1, TNF, TP53, IL6, and VEGFA. RT-qPCR validation confirmed the downregulation of inflammatory mediators like IL6 and PTGS2, alongside the upregulation of genes associated with potential cartilage repair effects, such as MMP1 and MMP3, upon HXZTS treatment. ConclusionsHXZTS exerts therapeutic effects on osteoarthritic synovial tissue through a multifaceted mechanism involving modulation of inflammation, oxidative stress, apoptosis, angiogenesis, and metabolic regulation. These findings provide insights into the potential application of HXZTS as a complementary or alternative therapeutic approach for managing OA and a foundation for developing novel targeted therapies.

bioinformatics↗

bioRxiv2025

XomicsDB: a database of paired multi-omics datasets for systems biology

Life science research is primarily driven by data integration on the basis of theoretical development. Broad applications of multiomics (xomics) data calls for efficient integration of xomics data to infer cellular operation and regulation. To that end, it is urgently needed good-quality xomics data should be curated and shared for the community. Here we present XomicsDB, a database curating paired xomics datasets at both bulk and single-cell levels in model organisms. It contains 183 datasets from 13 model organisms, with a total of seven xomics data types. Users can download these datasets individually or in a batch from the ftp server provided. Users can also upload their paired xomics datasets for sharing. Incoporation of xomics data intom GSMs are demonstrated in model organisms Escherichia coli and human. Future efforts will be on collecting more single-cell xomics data and curating non-model oragnisms. The XomicsDB can be found at: http://xomicsdb.com.

systems biology↗

bioRxiv2025

RRAS and RRAS2 mutations are oncogenic drivers in lung cancer and are sensitive to the pan-RAS inhibitor RMC-6236

IntroductionRRAS and RRAS2 encode a subfamily of RAS-like small GTPases that share considerable structural and functional similarities with KRAS, HRAS, and NRAS. Whether homologous RRAS/RRAS2 mutations are oncogenic and actionable drivers in lung cancer remains underexplored. MethodsAn institutional cohort of 8,488 non-small cell lung carcinomas (NSCLC) sequenced by comprehensive targeted DNA sequencing (MSK-IMPACT) between 2016-2024 was evaluated for RRAS/RRAS2 mutations. RRASQ87L or RRAS2Q72L were modeled in murine IL3-dependent Ba/F3 cells and immortalized human bronchiolar epithelial cells (HBECs). The oncogenic potential, signaling characteristics, and sensitivity to PI3K and MAPK pathway inhibitors, including the novel pan-RAS inhibitor RMC-6236, were evaluated in vitro and in vivo. ResultsRRASQ87L or RRAS2Q72L, homologous to KRAS-codon Q61 substitutions, were found in [~]0.45% of NSCLCs (38/8,488), with all but two lacking other MAPK pathway oncogenic drivers. RRASQ87L and RRAS2Q72L mutations transformed Ba/F3 and HBEC cells and robustly activated MAPK and PI3K-mTOR pathway signaling. RMC-6236 suppressed proliferation of RRASQ87L and RRAS2Q72L mutant cell lines, reduced ERK phosphorylation, induced apoptosis, and impeded cell-cycle progression. In vivo, RMC-6236 significantly inhibited growth of RRASQ87L/RRAS2Q72L-mutant HBEC-derived xenografts. ConclusionsRRASQ87L and RRAS2Q72L are recurrent, oncogenic, and potentially actionable drivers in NSCLC. Our study supports the inclusion of RRAS/RRAS2 into routine molecular diagnostic panels for precision oncology and provides preclinical rationale for investigating the potential therapeutic utility of pan-RAS inhibitors for patients with RRASQ87L/RRAS2Q72L-mutant lung cancers. Statement of translational relevanceTargeted therapies have transformed standard of care for oncogene-driven non-small cell lung carcinomas (NSCLC), yet a significant subset lacks actionable drivers. We identified recurrent RRASQ87L and RRAS2Q72L mutations which are mutually exclusive with other MAPK pathway drivers and found in [~]0.45% of NSCLC, comparable in prevalence to NTRK and NRG1 fusions. In preclinical models, these mutations activate canonical growth signaling, drive tumorigenic phenotypes, and confer sensitivity to RAS/MAPK-directed agents, including the novel pan-RAS inhibitor RMC-6236, currently in trials for patients with solid tumors harboring KRAS mutations. These data support RRASQ87L and RRAS2Q72L as bona fide lung cancer drivers and nominate RRAS/RRAS2-mutant tumors as candidates for pan-RAS-targeted therapeutics. Our findings provide a biologic rationale and preclinical evidence to inform molecular testing paradigms and to prioritize enrollment of patients with RRAS/RRAS2-mutant NSCLC into future clinical trials of pan-RAS inhibitors.

cancer biology↗

bioRxiv2025

PI-FC: Pre-training Individual-specific Functional Connectome through State-invariant Contrastive Learning

Functional MRI enables non-invasive mapping of brain connectivity, yet its clinical translation remains hindered by uncontrolled state-dependent variability that obscures individual-specific signatures during routine scanning. Here we introduce PI-FC -- a deep learning framework leveraging state-invariant contrastive learning to extract stable individual brain signatures across diverse arousal levels, cognitive states, and temporal scales spanning tens of seconds to hours. PI-FC achieves equivalent phenotypic prediction accuracy using substantially reduced scanning time, and eliminates state-dependent effects varying task demands and brain states. Trained on 36,119 subjects across 8 independent datasets, our model demonstrates superior cross-site generalization and outperforms traditional functional connectome (FC) in predicting neuropsychiatric conditions including schizophrenia, autism, depression, and anxiety. Furthermore, PI-FC enables zero-shot inference of brain age, biological sex, and cognitive ability without site-specific retraining. Overall, PI-FC represents a robust, clinically scalable framework that overcomes fundamental barriers to real-world deployment of precision functional neuroimaging.

neuroscience↗

bioRxiv2025

Fork Reversal Safeguards Epigenetic Inheritance during DNA Replication Under Stress

During DNA replication, epigenetic information carried by histone modifications is faithfully propagated and re-established on sister chromatids, ensuring cell identity. Chromatin reassembly is tightly coupled to DNA replication, therefore whether and how perturbations to DNA replication affects the fidelity of epigenetic inheritance remain poorly understood. In this study, we reveal a critical role for replication fork reversal in maintaining the transmission of epigenetic information under replication stress. We identify that cells defective in fork reversal exhibit reduced nucleosome density at replication forks, accompanied by the loss of parental histones during their transfer onto nascent DNA. Mechanistically, we demonstrate that PrimPol activation leads to single-stranded DNA gaps in fork reversal deficient cells, and that subsequent PARylation (poly ADP-ribosylation) and DNA-protein crosslinking on these gaps evicts nucleosomes. Our findings demonstrate that replication fork reversal, a widespread physiological process, is not only essential for preserving genome integrity but also for safeguarding epigenetic stability.

biochemistry↗

bioRxiv2025

Bodily Self in Macaque Monkeys and Human Infants

Whether monkeys possess the concept of bodily self remains controversial. We found that macaque monkeys trained for mirror self-recognition (MSR) could spontaneously recognize themselves when viewing video images of their back from a third-person perspective (3PP), but this generalization required a few minutes of "exploration". In contrast, through visual-tactile synchronization training similar to that used in human out-of-body studies, naive monkeys could recognize themselves from 3PP back images and demonstrated MSR within seconds. Interestingly, in human infants, we found that 3PP back self-recognition developed spontaneously several months after MSR. These results demonstrate the existence of hierarchical bodily self in macaques and similar stepwise development of bodily self in human infants, offering insights into the emergence and manifestation of bodily self in natural and artificial intelligence.

neuroscience↗

bioRxiv2025

The role of miR-9a in modulating sensory neuron morphology and mating behavior in Drosophila melanogaster.

Following mating, female Drosophila melanogaster display profound behavioral changes, including sensory sensitivity and rejection of courting males. The molecular mechanisms governing this plasticity remain incompletely understood. Here, we identify the conserved microRNA, miR-9a, as a critical regulator of this process. We show that miR-9a mutant females exhibit a premature rejection phenotype, mimicking mated-female behavior, which is correlated with an aberrant overgrowth of adult body wall sensory neurons. We demonstrate that this neuronal phenotype is governed by a dual regulatory system. First, in a non-cell autonomous mechanism, miR-9a expression in the epidermis is required to constrain sensory neuron dendrite growth, indicating that an epithelial-derived signal patterns the underlying neuron. Second, within the neuron itself, miR-9a interacts genetically with the transcription factor senseless (sens) and the novel RNA-binding protein bruno2 (bru2). Reducing the dosage of either sens or bru2 rescues both the neuronal and behavioral defects of miR-9a mutants. Our findings reveal an integrated, inter-tissue signaling axis where epithelial miR-9a orchestrates a non-cell autonomous cue that modulates a cell-intrinsic network to ensure the precise development of sensory neurons, thereby calibrating behavioral responses critical for reproductive success.

neuroscience↗

bioRxiv2025

Dual checkpoint blockade of glioblastoma with Anti-PD-1 and Anti-LAG-3 promotes expansion of tumor-reactive T cell clones along a unique pathway of differentiation

IDH-wildtype grade IV glioblastoma is the most aggressive adult primary brain tumor and remains refractory to anti-PD-1 monotherapy despite evidence of limited tumor-specific T cell induction. To determine the impact of immune checkpoint inhibitors (ICIs) on glioblastoma T cell transcriptional landscape and repertoire, we conducted paired single-cell RNA sequencing (scRNA-seq) and T cell receptor sequencing (TCR-seq) of tumor-infiltrating lymphocytes (TILs) from patients with untreated, newly diagnosed glioblastoma and from recurrent glioblastoma treated with dual checkpoint blockade targeting PD-1 and LAG-3. Using a validated transcriptional signature, we found that predicted tumor-reactive T cells (TRC) in untreated glioblastomas reside almost exclusively in a clonally expanded GZMKhi population with developmental plasticity, affording them the potential to differentiate into both tissue-resident and terminal effector T cells. Dual ICI therapy induced substantial clonal remodeling, characterized by the recruitment of new TRC from the periphery into the tumor microenvironment (TME) and differentiation into transitional effectors and ultimately terminal effectors along a gradient characterized by simultaneous acquisition of cytotoxic and exhaustion genes, regulated by specific transcriptional, metabolic, and epigenetic programs. Longitudinal clonal tracking in peripheral blood confirmed that with ICI treatment, most TRC expand transiently in circulation prior to tumor infiltration, with peripherally derived clones becoming the major contributor to the GZMKhi TRC that further expand in the tumor. Our study provides the first comprehensive map of T cell clonal dynamics and differentiation in glioblastoma following dual ICIs and highlights a potential mechanism of immune activation and peripheral recruitment of TRC in glioblastoma not previously described. Our results suggest that therapeutic strategies to sustain these GZMKhi early effector and transitional effector T cells may further enhance ICI therapeutic efficacy in glioblastoma.

immunology↗

bioRxiv2025

Increasing Applicability of Automated mD-LC-MS Peptide Mapping for Biopharmaceuticals via Streamlined In-Solution Digestion

Since the introduction of the first multidimensional liquid chromatography mass spectrometry (mD-LC-MS) approach for antibody peak characterization, multiple developments have been reported including integration of various chromatographic approaches and precise fractionation, providing high quality and reliable peptide data in a drastically decreased total analysis time. Most of these platforms rely on the use of immobilized enzyme reactors (IMERs) for digestion, limiting the applicability to few enzymes such as trypsin. Recently, the introduction of in-solution enzymatic digestion in mD-LC-MS systems has been proposed as an alternative to IMERs. Here, we make use of current innovations in 2D-LC commercial systems such as active solvent modulation valves, which permits direct mixing of the fractionated peaks with the endoprotease and reducing agent in an online manner, to integrate in-solution digestion in a straightforward manner in mD-LC-MS peak characterization platforms. Following antibody digestion, the generated peptides are automatically trapped, separated and detected by mass spectrometry. Efficient reduction and tryptic digestion was obtained using short incubation times (15 min). The approach was further expanded to alternative endoproteases such as chymotrypsin and thermolysine with other digestion specificities and allow an easy exchange between enzymes with similar buffer and digestion conditions. As a proof-of-principle that strategy was applied to achieve peptide maps from ion exchanged separated mAb peaks showing good digestion efficiency and high sequence coverages.

biochemistry↗