Biology subjects

Acharya, S.

Publications and source records attributed to Acharya, S..

At least 19 recordsLinked to original sources

bioRxiv2026

SPARK: Bayesian structure learning of enhancer-based gene regulatory networks from single-cell multiomics data and prior knowledge

Enhancer-based gene regulatory networks (eGRNs) linking enhancers with recruited transcription factors (TFs) and downstream target genes can help decipher complex regulatory mechanisms, such as the function of GWAS variants at enhancers. Molecular assays that profile gene expression and chromatin state jointly from the same cells (such as 10x Multiome) are a rich source of input data for eGRN reconstruction. However, these assays lack mechanistic evidence for enhancer-gene and TF-enhancer links, which can be contributed by prior knowledge from other assays and analyses. Here, we present SPARK, an eGRN reconstruction framework that formally combines single-cell multiome data with mechanistic prior knowledge. SPARK represents eGRNs as directed acyclic graphs (DAGs) and learns them using Bayesian inference, with a prior on the network structure constructed from heterogeneous sources of evidence. In a simulation study, we show that SPARK benefits from prior knowledge and outperforms existing DAG-based and regression-based algorithms. Finally, we use SPARK to study the regulatory circuitry of enhancers harboring immune disease GWAS variants in human primary monocytes, revealing known and novel regulatory links as candidates for experimental validation.

genomics↗

bioRxiv2026

Mitochondrial and metabolic plasticity distinguish stem cells from progenitors in the male germline

Metabolism and mitochondrial remodelling are increasingly recognised as key regulators of stem cell identity, governing the balance between self-renewal and differentiation. Here we compared mitochondrial and metabolic properties of stem and progenitor cells from the male germline using transmission electron microscopy, quantitative proteomics and extracellular flux analysis. While the stem cell-to-progenitor transition was accompanied by increased mitochondrial abundance and cristae density, our analyses unveiled a remarkable and unexpected capacity for spermatogonial stem cells (SSCs) to engage oxidative phosphorylation. When oxygen availability was unrestricted, SSCs displayed markedly greater maximal and spare respiratory capacity than progenitors, despite comparable glycolytic flux. SSCs were also enriched for glutathione-dependent antioxidant enzymes and maintained lower basal mitochondrial superoxide levels than progenitors. Collectively, these findings challenge the view that SSCs are predominantly glycolytic and instead identify metabolic plasticity, marked by latent oxidative capacity and enhanced redox control, as a hallmark of the stem cell state.

cell biology↗

bioRxiv2026

Genetic and structural interpretation of NLRP1 FIIND domain variants in glioma in an Indian cohort: a pilot study

Gliomas, particularly glioblastoma (GBM), are aggressive primary brain tumors associated with dysregulated NLR signaling, a pathway central to innate immunity and inflammation. NLRP1 triggers proinflammatory cytokine release and pyroptotic cell death via autoproteolytic cleavage. The FIIND missense variant rs11651270 (M1184V) may modulate this cleavage process. While NLRP1 polymorphisms are associated with various diseases and cancers, their specific impact on glioma remains to be investigated. In our study, five FIIND-domain single-nucleotide polymorphisms (SNPs) of NLRP1 rs371579423, rs58604457, rs57636751, rs11651270, and rs2301583, were investigated by Sanger sequencing in a clinical cohort of glioma patients and compared against population-matched controls from the GenomeIndia dataset (Rajasthan cohort) using genetic association models. Molecular dynamics simulations were performed to evaluate the structural effects of the missense variant rs11651270 (M1184V) in NLRP1 during pre- and post-cleavage states. The linked variants rs58604457 (G>A) and rs57636751 (C>T) exhibited complete linkage disequilibrium (r2=1.00) and were significantly associated with lower odds of glioma (odds ratio ~0.5). The missense variant rs11651270 (T>C) showed no association with glioma risk. Notably, the SNP rs2301583 had a higher allelic frequency in glioma cases despite being absent in the GenomeIndia population-based control catalogue. Molecular dynamics simulations revealed that the M1184V substitution stabilizes local FIIND architecture by preserving {beta}-strand organization through persistent interactions with neighboring residues. This study provides the first combined genetic and structural analysis of NLRP1 FIIND-domain variants in an Indian glioma cohort. These findings illustrate the potential value of integrating population-based genetic association with structural modelling to generate hypotheses and uncover potential functional mechanisms of inflammasome-gene variants in neuro-oncology.

cancer biology↗

bioRxiv2026

Protein size and geometry govern mutational robustness

A fundamental question in structural biology centres around understanding protein evolution. Key to this process is mutational robustness, defined as the protein fold's ability to absorb sequence changes without collapsing its structure. Here, we show that robustness is systematically shaped by simple features such as protein size, geometry, and oligomeric state. We used Foldseek-identified (structural) homologs to quantify family size across monomers and higher homo-oligomers. We found that proteins in larger families are consistently larger in size, more compact in atomic density, and less exposed to solvent. Strikingly, homo-oligomers occupy systematically larger families than monomers, revealing quaternary structure itself as a driver of mutational tolerance, not merely a functional supplement. This signature of robustness can be further linked to increasing functional complexity in proteins; those with adaptive, multifaceted biological roles belong to larger structural families than those with specific roles, thereby linking structural flexibility directly to evolutionary versatility. In short, simple yet overlooked features of protein geometry can explain mutational robustness and evolvability, offering a structural rationale for why certain protein families have diversified extensively while others remain in evolutionary stasis.

bioinformatics↗

bioRxiv2026

TREM2 drives monocyte-derived macrophage responses to Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungus that causes pulmonary and central nervous system infections after entry into the lungs. We previously established that signaling through the adapter protein DAP12 inhibits the antifungal response of monocyte-derived macrophages and worsens the survival of mice after C. neoformans infection. However, the molecular mechanisms by which DAP12 signaling is initiated during cryptococcosis remain inadequately characterized. In this study, we identify triggering receptor expressed on myeloid cells 2 (TREM2) as a DAP12-associated receptor that is induced on murine monocytes and interstitial macrophages in the lungs in response to C. neoformans infection. TREM2 subsequently represses fungal uptake and M1 polarization by monocyte-derived macrophages. Using an in vitro binding assay, we find that both murine and human TREM2 can directly bind to C. neoformans and that the absence of the cryptococcal cell wall antigen {beta}-1,6-glucan disrupts these interactions. Overall, our findings suggest that the TREM2-DAP12 pathway plays an important inhibitory role in the host immune response to cryptococcal infection by impeding macrophage activation and phagocytosis of fungal cells. We also establish TREM2 as a receptor involved in direct fungal sensing of C. neoformans.

immunology↗

bioRxiv2026

Proton FLASH preserves neurocognition across delivery techniques: implications for clinical translation in pediatric brain tumors

BackgroundRadiation therapy is integral to the curative treatment of childhood brain tumors but contributes to late neurocognitive impairment in survivors. FLASH (ultra-high dose rate, >40Gy/s) reduces normal-tissue toxicity in preclinical models, and proton-FLASH is currently the only modality capable of delivering ultra-high dose rates to the deep targets, such as pediatric brain tumors. However, two questions remain unresolved before clinical translation: (1) whether the FLASH effect can be achieved on synchrotron-based proton systems, which deliver protons in discrete spills that may be insufficient to cover a clinical target within a single delivery, and (2) which dose-rate metric, among the multiple definitions currently used in the field, best predicts the biological FLASH effect. MethodsC57BL/6 mice (7-8 weeks) received 10 Gy whole-brain RT via a clinical Hitachi ProBEAT synchrotron with CBCT-guided delivery, using three transmission-beam techniques: single-spill pencil beam scanning (SS PBS), multi-spill PBS with [~]2-second inter-spot delay (MS PBS), and passive scatter (PS), compared to conventional (CONV) delivery and unirradiated controls (n=24-28/group, equal sex distribution). Dose rate was quantified using three frameworks: field dose rate (FDR), PBS dose rate (PBSDR), and dose-averaged dose rate (DADR). Recognition memory was assessed by novel object recognition (NOR) at 6 weeks post-RT, and cognitive flexibility was assessed via touchscreen visual discrimination and reversal learning at 14 weeks. Hippocampal neuroinflammation was evaluated by immunofluorescence and immunohistochemistry for Iba1, NeuN, and GFAP. ResultsFLASH conditions were met by SS PBS and PS under all three dose-rate definitions, but MS PBS qualified as FLASH only by DADR. Despite this, neuroprotection was preserved across all three FLASH techniques: discrimination index was significantly higher for SS PBS (P=0.021), MS PBS (P=0.008), and PS (P<0.001) versus CONV, with no significant difference between FLASH techniques. On touchscreen testing, FLASH-treated females demonstrated preserved cognitive flexibility (P=0.047 vs. CONV on reversal learning correct trials). Iba1+ microglia were reduced in FLASH compared to CONV mice, with morphology suggestive of preserved homeostatic state. ConclusionsSynchrotron-based proton FLASH preserves neurocognitive function across all delivery techniques, including under multi-spill delivery essential for treating clinical-scale pediatric brain tumors. Critically, this neuroprotection was observed even for deliveries that qualified as FLASH only by DADR, identifying DADR as the dose-rate metric most relevant to the biological FLASH effect with direct implications for clinical trial design and dose-rate reporting standards.

cancer biology↗

bioRxiv2026

Sympathetic neurons control adaptive immunity in response to Streptococcus pneumoniae infection by regulating T cell and B cell effector function

Adaptive immunity in the lung must be rapidly mobilized to control bacterial pathogens, yet the neuroimmune signals that shape local B and T cell responses remain poorly defined. Here we identify a sympathetic-immune axis that regulates lung-resident memory B cells, antigen-specific IgG production, and protection against Streptococcus pneumoniae. Lung-targeted chemical sympathectomy reduced norepinephrine availability, diminished Tbet memory and resident-memory B cell populations, and markedly impaired S. pneumoniae-specific IgG, resulting in increased bacterial burden. Mice lacking Adrb1 & Adrb2-adrenergic receptors phenocopied these defects, and adoptive transfer of Adrb1/Adrb2-/- B cells into Ighm-/- hosts was sufficient to reduce IgG and worsen infection. Combined T cell depletion and B cell-intrinsic adrenergic receptor loss further impaired bacterial control and suppressed IFNgamma, indicating that norepinephrine stimulates T cell IFNgamma release and that norepinephrine and IFNgamma together drive optimal B cell IgG production. In vitro, norepinephrine enhanced T cell IFNgamma secretion, and norepinephrine plus IFNgamma synergistically increased IgG. These findings define norepinephrine as a key regulator of lung-resident humoral immunity and reveal a neuroimmune mechanism that promotes antibody-mediated bacterial clearance at the respiratory mucosa.

immunology↗

bioRxiv2026

Multimodal antioxidant and anti-inflammatory activity of Rauwolfia serpentina root extracts in experimental models

BackgroundChronic inflammation and oxidative stress are central drivers of cardiovascular disease progression and remain incompletely addressed by existing pharmacological strategies. Traditional medicinal plants provide a valuable source of multi-target bioactive compounds that may modulate these interconnected pathways. Rauwolfia serpentina, a classical antihypertensive plant in Ayurveda, has been historically valued for cardiovascular indications. Yet, its antioxidant and anti-inflammatory actions beyond blood pressure regulation remain insufficiently characterised in immune-driven inflammatory models. MethodsRoot extracts of R. serpentina prepared using hot and cold ethanol and water were evaluated for antioxidant capacity using DPPH radical scavenging and phosphomolybdenum assays, along with phenolic, flavonoid, and terpenoid quantification. Protective effects against lipid peroxidation were assessed in rat liver and heart homogenates. Anti-inflammatory activity was examined in THP-1 human monocytic cells exposed to lipopolysaccharide (LPS), arachidonic acid (AA), and oxidative stress. Cytokine secretion and gene expression of TNF-, MCP-1, IL-6, and IL-8 were measured by ELISA and qRT-PCR. Intracellular reactive oxygen species and catalase activity were quantified to assess oxidative regulation. LC-MS-based metabolomic profiling was performed to characterise chemical diversity. The principal alkaloid, reserpine, was evaluated separately, and molecular docking was performed to examine its interaction with IKK. ResultsEthanolic extracts of R. serpentinas root, particularly the cold ethanolic fraction, showed superior antioxidant capacity, higher phenolic and flavonoid content, and potent inhibition of lipid peroxidation. These extracts markedly suppressed LPS-induced cytokine release and gene expression in THP-1 cells, with pronounced effects on MCP-1 and IL-6. Oxidative stress induced by arachidonic acid was attenuated through reduced intracellular ROS and preservation of catalase activity. Reserpine reproduced key features of the extract response, demonstrating strong suppression of IL-6 and MCP-1 at both transcriptional and secretory levels. Docking analysis indicated stable binding of reserpine within the IKK catalytic pocket, supporting a plausible mechanism for modulation of the NF-{kappa}B pathway. ConclusionR. serpentina root extracts exhibit coordinated antioxidant and anti-inflammatory activity in immune cell models relevant to cardiovascular inflammation. These effects are extraction-dependent and are partially mediated by reserpine through modulation of oxidative stress and inflammatory signalling pathways. The findings support the translational relevance of R. serpentina as a traditional medicine with mechanistic activity extending beyond antihypertensive action.

plant biology↗

bioRxiv2026

MassID provides near complete annotation of metabolomics data with identification probabilities

Liquid chromatography coupled to mass spectrometry (LC/MS) is a powerful tool in metabolomics research, generating tens-of-thousands of signals from a single biological sample. However, current software solutions for unbiased assessment of metabolomics data analysis are limited by complex sources of noise and non-quantitative metabolite identifications that make results difficult to interpret. Here, we present MassID, a cloud-based untargeted metabolomics pipeline that aims to overcome the innate challenges of unbiased metabolite analysis and perform end-to-end data processing, transforming raw spectra to normalized and identified metabolite profiles. MassID incorporates a suite of software functionalities, including deep learning-based peak detection and comprehensive noise filtering. In addition, with MassID we introduce a novel software module: DecoID2 that enables probabilistic metabolite identification for false discovery rate (FDR)-controlled metabolomics. When applied to a human plasma dataset, MassID results in near-complete signal annotation, identification of >4,000 metabolites (including >1,200 compounds at an FDR <5%) across four complementary LC/MS runs, and enables integrated downstream analyses to understand biochemical dysregulation at both the molecular and pathway level. When compared to the Metabolomics Standards Initiative (MSI) confidence levels, identification probability generally correlated with MSI levels. However, only 356/418 of MSI Level 1 compounds were identified with <5% FDR and the remaining 884 FDR < 5% compounds were identified from MSI L2-L3 compounds, highlighting the enhanced specificity and discovery potential achieved by MassID.

bioinformatics↗

bioRxiv2026

ST2 Signaling Regulates Innate Immune Responses in Kidney Injury

IntroductionInnate immune cells are critical in inflammation, repair, and fibrosis post-kidney injury. Nuclear-cytokine interleukin (IL)-33, which is released upon tissue damage, signals through IL-1-receptor-like-1 (IL1RL1 or ST2), expressed on many immune cells, including macrophages. However, macrophage regulation by IL-33/ST2 is incompletely understood. We hypothesized that ST2 plays a vital role in activating and/or mobilizing myeloid cells and macrophages to sites of injury. MethodsWe performed acute and chronic ischemia-reperfusion injury (IRI) in mice with myeloid cell-specific deletion of ST2 (ST2fl/fl.LysMCre) to examine the role of myeloid cells ST2 expression in renal injury. The structure and function of the kidney were probed using flow cytometry, histology, immunohistochemistry, quantitative gene expression, and biochemical analysis. The invitro efferocytosis assay, RNA Seq, and Seahorse assay were carried out using bone-marrow-derived macrophages ResultsInterestingly, ST2 deletion resulted in attenuated renal pathology in the acute renal IRI model, whereas in chronic IRI, the loss of ST2 exacerbated kidney injury, suggesting a role of ST2 in the resolution of chronic injury. RNA sequencing (RNASeq) analysis of bone-marrow-derived ST2 sufficient and deficient macrophages showed that loss of ST2 downregulated genes involved in oxidative phosphorylation and clearance of dead cells (efferocytosis). Indeed, the ST2-deficient macrophages had reduced phagocytosis activity. Further, Seahorse analysis revealed that ST2-deficient macrophages had compromised mitochondrial metabolism. ConclusionsWe conclude that the IL-33/ST2 axis is essential for regulating macrophage function and contributes to regulating tissue homeostasis following renal injury.

immunology↗

bioRxiv2026

Integrative analysis of Myrcene's lipase inhibition and anti-inflammatory mechanisms in human monocytic cells

Myrcene (MCN), a monoterpene commonly present in essential oils of aromatic plants, was investigated for its dual role in modulating lipid metabolism and inflammation in human monocytic THP-1 cells. MCN was first evaluated for its ability to inhibit pancreatic lipase activity, followed by cytotoxicity screening to determine non-toxic working concentrations. Its anti-inflammatory effects were assessed by measuring TNF- gene expression and secretion in LPS-stimulated cells using qRT-PCR and ELISA, while NF-{kappa}B p65 nuclear translocation was examined through immunofluorescence. The influence of MCN on macrophage differentiation was further determined in PMA-treated THP-1 cells. MCN significantly reduced pancreatic lipase activity and suppressed inflammatory responses by downregulating TNF-, TLR2, and MIP-1, alongside preventing NF-{kappa}B p65 nuclear migration. Molecular docking supported these findings by confirming favorable interactions of MCN with the catalytic pocket of lipase and the nuclear localization region of NF-{kappa}B. Collectively, this study identifies MCN as a promising multitarget natural compound with potential therapeutic application in metabolic and inflammation-associated disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/699177v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@404f01org.highwire.dtl.DTLVardef@2e67aorg.highwire.dtl.DTLVardef@697050org.highwire.dtl.DTLVardef@4cb84d_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Proteus mirabilis entails a switch from commensal to pathogen - Genomic insights from a blaTEM-1B-harboring novel isolate from India.

BackgroundProteus mirabilis is an opportunistic pathogen that can shift from gut commensalism to causing severe urinary tract and bloodstream infections. The genomic basis of P. mirabilis transition from commensalism to severe infection is not well understood, especially in Indian clinical isolates. This study aimed to explore the genomic attributes of a prevalent P. mirabilis strain in India. MethodsWhole-genome sequencing (WGS) was performed on a clinical P. mirabilis isolate by using NGS. AMR genes and virulence factors were identified using ResFinder/CARD and VFDB, respectively. Sequence typing was performed with MLST. Protein-protein interactions and noncoding RNAs were analyzed using STRING, CytoHubba, and BAKTA. ResultsWGS confirmed the multidrug-resistant nature of the isolate, revealing a broad repertoire of AMR genes, including blaTEM-1B, conferring resistance to beta-lactam antibiotics. Several resistance determinants were co-localised with insertion sequences on a single contig, indicating a high potential for horizontal gene transfer. The virulome profile highlighted multiple fimbrial genes, hemolysins, and toxin-encoding loci, together supporting strong adhesion, biofilm formation, and host tissue damage. Comparative analysis revealed unique genomic signatures not present in reference isolates, including additional virulence determinants and mobile genetic elements. MLST profile of the isolate is closely related to ST-675 & ST-797, while virulence typing (vST-1157) indicated a novel profile distinct from reference ST-675 strains. Insilico protein-protein interaction of this isolate revealed a highly interconnected virulence network, with urease, fimbrial adhesins, and hemolysins emerging as central functional modules driving pathogenicity. In addition, a noncoding RNA was identified within the genomic context of the ure operon (Contig 10), suggesting a possible regulatory role in urease expression. PPI network analysis highlighted urease, fimbrial proteins, and hemolysins as central hubs contributing to pathogenicity. ConclusionsThis study presents the first genomic characterization of a multidrug-resistant P. mirabilis clinical isolate from India, designated as P. mirabilis strain Indica. The convergence of AMR genes, virulence factors, MGEs and a novel noncoding RNAs linked to urease expression highlights the emergence of a high-risk variant in India. These findings have significant implications for infection control and emphasize the need for genomic surveillance to guide therapeutic strategies and develop new interventions globally.

microbiology↗

bioRxiv2025

Cation-exchange synthesized Zn-doped Ag2S Nanostructures for Photothermal and Photodynamic Therapies across Breast Cancer Subtypes

Photothermal therapy (PTT) and photodynamic therapy (PDT) require nanostructures capable of efficiently converting red-light energy into both heat and reactive oxygen species (ROS). However, simultaneously achieving high photothermal conversion efficiency and strong ROS generation remains challenging. Here, we report zinc-doped Ag2S (ZSS) nanostructures, synthesized via controlled cation exchange, in which Zn incorporation modulates the electronic structure of Ag2S and improves charge separation, thereby enhancing red-light-activated PTT/PDT performance while preserving intrinsic biocompatibility. Among the series, ZSS(0.15) demonstrated optimized charge-carrier dynamics, a high photothermal conversion efficiency of 67.26%, and approximately four-fold higher singlet oxygen (1O2) generation relative to methylene blue under 660 nm irradiation. These physicochemical enhancements translated into potent therapeutic outcomes: in-vitro, ZSS(0.15) achieved an IC50 of 15 {micro}g/mL under irradiation, corresponding to approximately a 1.5-fold enhancement in cytotoxic potency compared to pristine Ag2S, and induced apoptosis via activation of the p53/Bax/Caspase pathway. In-vivo, ZSS(0.15) with laser irradiation achieved [~]97% tumor volume suppression without systemic toxicity. Extending evaluation across multiple breast cancer cell lines representing distinct molecular subtypes further confirmed broad-spectrum in-vitro efficacy. Altogether, these findings demonstrate that controlled Zn incorporation into Ag2S effectively enhances red-light-driven photothermal conversion and 1O2 generation, establishing a rational materials strategy for improving synergistic PTT/PDT performance. GRAPHICAL ABSTRACTA Zn-doped Ag2S nanoplatform (ZSS(0.15)) enables synergistic photothermal and photodynamic therapy under 660-nm laser irradiation, triggering p53-mediated mitochondrial apoptosis via ROS generation and caspase-3 activation, achieving [~]97% tumor regression in-vivo without detectable systemic toxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/684667v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1d93519org.highwire.dtl.DTLVardef@12d18f4org.highwire.dtl.DTLVardef@1c14608org.highwire.dtl.DTLVardef@1dcd9f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Combining Motifs, CRE Activity, And Gene Expression Data Using ML Greatly Improves the Accuracy of Tissue-Specific TF Network Maps

MotivationReconstructing tissue-specific transcription factor (TF) networks remains challenging. TF motif-based methods often lack functional validation, while expression-based methods struggle to distinguish direct binding from indirect regulation. Integration of diverse data types is necessary to accurately prioritize functional targets directly bound by TFs across human tissues. ResultsWe introduce METANet, a supervised ensemble learning framework that combines TF motifs, cis-regulatory element activity, and linear and non-linear expression-derived features to predict TF binding. Applied to 36 human tissues, METANet significantly outperforms established methods in identifying direct, functional targets of TFs validated by ChIP-seq and gene ontology. Furthermore, METANet captures tissue-specific regulation comparable to existing methods, allowing the identification of reproducible gene-trait associations. Availability and ImplementationAll code and network maps are freely available at Zenodo https://doi.org/10.5281/zenodo.17309371. Contactbrent@wustl.edu.

systems biology↗

bioRxiv2025

LoAR-Exo and HiAR-Exo: One-step label-free isolation of extracellular vesicles using inertial microfluidic devices

Extracellular vesicles (EVs) are lipid membrane-bound nanoscale (20 nm - 1000 nm) objects shed by all cells. As EVs play an important role in intercellular signaling, these have emerged as promising biomarkers for many diseases including cancer and neurodegenerative disorders. A major bottleneck in research into EVs and their smooth translation to clinic as disease biomarkers is a lack of access to affordable and user-friendly technologies to quickly isolate EVs from complex biological and clinical samples with acceptable purity and yield. Here, we report two different designs of inertial microfluidic devices (e.g. LoAR-Exo and HiAR-Exo) that can isolate EVs from different kinds of biological samples in a single label-free step using a spiral microchannel design with different aspect ratios. LoAR-Exo has a height-to-width aspect ratio [~] 1, while HiAR-Exo has a height-to-width aspect ratio of 2.5, optimized by COMSOL simulations. We tested both designs by separating polystyrene particles of size < 1 m from a heterogeneous mixture. We then benchmarked the performance of the microfluidic chips against ultracentrifugation and a precipitation kit by isolating EVs from the cell culture-conditioned media (CCM) of MDA-MB-231 cells. We also compared the microchip with SEC by using the CCM from H1975/OR cells. The size distribution of EVs isolated by the microfluidic chip was comparable with ultracentrifugation, precipitation and SEC. In summary, both devices isolated EVs from as little as 1 mL of sample volume using a label-free technique in a continuous manner and without any user intervention. Both microfluidic platforms offer a simple, efficient, and scalable alternative to conventional methods for EV isolation.

bioengineering↗

bioRxiv2025

GM-CSF orchestrates monocyte and granulocyte responses to Cryptococcus gattii

Cryptococcus gattii is an emerging fungal pathogen that is acquired through the respiratory tract and causes invasive infections in both immunocompromised and otherwise healthy people. Many of these apparently immunocompetent patients are subsequently found to have autoantibodies against the pleiotropic cytokine GM-CSF. In this study, we investigated the potential role of GM-CSF (or CSF2) in the host response to C. gattii using a murine model of infection. Interestingly, infected Csf2-/-mice were found to have significantly improved survival and decreased lung fungal burden compared to wild type (WT) mice. We determined that during C. gattii infection, GM- CSF promotes the differentiation of monocytes into alveolar and interstitial macrophages. When these macrophages are ablated in CCR2-DTR+ mice, there is a corresponding improvement in survival with decreased lung fungal burden, thus phenocopying Csf2-/- mice. WT bone marrow-derived macrophages challenged with C. gattii have poor antifungal activity, suggesting that monocyte-derived macrophages (moMacs) are rendered permissive for fungal proliferation. Therefore, GM-CSF and moMacs mediate immune responses that are harmful to the host. We further found that GM-CSF and moMacs preferentially promote the influx of eosinophils over neutrophils into the infected lung. This eosinophilia is associated with substantial inflammatory lung pathology. Ablation of neutrophils using Mrp8cretg iDTR+ mice significantly increased C. gattii burden in the lungs, indicating that GM-CSF and moMacs block the entry of these beneficial, fungal-clearing granulocytes during infection. Altogether, our results show that GM-CSF plays a key role in impeding host anti-fungal responses to C. gattii by coordinating monocyte-derived macrophages and granulocyte activity and crosstalk. Author SummaryCryptococcus gattii is an environmental fungus that can cause severe lung and brain infections after inhalation through the respiratory tract. C. gattii causes disease in patients with known immune deficits but also in individuals that are apparently healthy. Studies on otherwise healthy people who become infected with C. gattii suggest that they may have a previously unrecognized problem involving granulocyte macrophage-colony stimulating factor (GM-CSF), a cytokine, or messenger protein, that is an important part of the immune system. Here, we investigate the role of GM-CSF in the immune response to C. gattii using a mouse model of infection. We find that C. gattii increases GM-CSF in the lungs, leading to the influx of immune cells, including monocyte-derived macrophages and eosinophils, while inhibiting the entry of neutrophils. The macrophages and eosinophils allow the fungus to proliferate and cause inflammatory damage to the lungs, which is ultimately fatal. The absence of neutrophils also contributes to fungal growth, as these immune cells would otherwise be able to help kill the fungus. Our study provides new insight into how GM-CSF regulates immunity to C. gattii and has important implications as to the mechanisms that govern susceptibility to this infection.

immunology↗

bioRxiv2025

Whole blood transcriptional signatures of age and survival identified in Long Life Family and Integrative Longevity Omics Studies

Although aging is a universal event, some individuals are able to achieve extreme longevity. The Long-Life Family Study (LLFS) enrolls participants from families enriched with long-lived individuals, serves as a valuable dataset for studying ageing phenotypes and identify potential intervention targets. We analyzed the association between age at blood draw and 16,284 RNAseq-based blood transcriptomic data from 2,167 LLFS participants with ages ranging from 18 to 107, replicated the results in the Integrative Longevity Omics Study (ILO) dataset of 20,884 RNAseq-based blood transcriptomic data from 419 participants, with ages ranging from 60 to 108, and further compared our findings to a published reference aging signature. We identified 4,227 transcripts increasing and 4,044 transcripts decreasing with age, and enrichment analysis revealed age-related upregulation of inflammatory and senescence-related pathways, and downregulation of MYC and Wnt/{beta}-catenin targets, among others. Further, a subset of transcripts showed age associations unique to the longevity-enriched cohorts (LLFS and ILO). We also identified 314 transcripts significantly associated with mortality risk and found that pro-survival gene sets included NK cell-mediated cytotoxicity and GPCR signaling. Finally, increased transcriptomic age predicted using transcriptomic clock was strongly associated with increased mortality. In summary, this study identified robust transcriptomic signatures of aging and mortality in a longevity-enriched population, highlighting key biological pathways such as immune modulation, inflammation, and senescence. Authors notesThis manuscript has been peer-reviewed and accepted by GeroScience (Springer). This bioRxiv article reflects the published version, incorporating revisions made in response to reviewers comments. The main content, results, and conclusions remain unchanged from the previous version, while the Discussion section has been expanded to further address the functional annotation and clinical relevance of the identified markers. Copy of the acceptance letterThe editors are pleased to inform you that your manuscript, JAAA-D-25-01946R1 entitled "Whole blood transcriptional signatures of age and survival identified in Long Life Family and Integrative Longevity Omics Studies" has been accepted for publication in GeroScience as an Original Article. The editors commend you on your outstanding contribution to the journal. Your manuscript can be published online ahead of print within approximately two weeks.

molecular biology↗

bioRxiv2025

Identification of novel protective loci for executive function using the trail making test part B in the Long Life Family Study

The Trail Making Test (TMT) Part B (TMT-B), a well-established assessment of cognitive function, is a frequent component of diagnostic assessments for Mild Cognitive Impairment and dementia in older adults. Identifying the genetic variants associated with the TMT-B will not only gain insights of genetic determinants of cognitive function, but also the molecular mechanisms for dementia. Published GWAS to date for TMT-B suffer from relatively low power due to the use of population level data and imputation methods. To address these deficits, we used a family-based study design to identify the genetic variants associated with the TMT-B incorporating both genome-wide linkage analysis (GWLS) and whole genome sequencing (WGS). As such, we examined the sequenced genetic determinants of TMT-B using GWLS in over 2000 participants from Long Life Family Study (LLFS). In GWLS, the estimated heritability of TMT-B was 0.29. We detected one significant linkage peak at 15q25 (LOD>3.0). Statistical fine-mapping nominated five variants including three SNPs (NTRK3-rs74031103, protective CEMIP-rs2271159, and protective AGBL1-rs4134376) and two INDELs (protective KLHL25-15:85882445:IND, and protective CEMIP-15:80893381:IND) contributing to the linkage peak. Four out of these five variants are protective for TMT-B. The rs2271159 SNP influences CEMIP expression in cerebellum and hippocampus, while the 15:80893381:IND modulates CEMIP expression in blood. Additionally, the variant rs4134376 is a basal ganglia-specific eQTL for AGBL1. In conclusion, we utilized GWLS, leveraged multi-omics data (whole genome sequence genomic data, transcriptomic data, and lipidomic data), and identified novel protective variants and genes for TMT-B performance.

genomics↗