Biology subjects

Chan, A.

Publications and source records attributed to Chan, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Localized SARS-CoV-2 Infection Triggers a Tissue-Wide Antiviral Response and Functional Impairment of Olfactory Sensory Neurons

Olfactory dysfunction is a hallmark of COVID-19, yet the mechanisms by which severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes widespread sensory impairment remain incompletely understood. We compared the olfactory epithelial tropism of multiple SARS-CoV-2 variants in a mouse model and observed that Alpha and Beta variants exhibited the greatest infectivity for the olfactory epithelium (OE), whereas Omicron rarely infected this tissue despite comparable pulmonary viral titers. We further defined the effects of SARS-CoV-2 on olfactory sensory neurons (OSNs) using immunohistochemistry, single-cell RNA sequencing, spatial gene expression characterization, and functional odor stimulation. The localized infection of sustentacular cells triggered a tissue-wide interferon-stimulated antiviral response that extended beyond infected regions for Alpha and Beta but was largely absent following Omicron infection. Mature OSNs transiently adopted an interferon-responsive state before exhibiting persistent downregulation of odorant signal transduction, mitochondrial, and activity-dependent gene pathways. Consistent with these transcriptional changes, odor stimulation failed to elicit normal activity-dependent gene expression during SARS-CoV-2 infection, indicating impaired neuronal function without widespread neuronal loss. Progressive accumulation of macrophages further indicated sustained inflammatory remodeling of the OE. Together, these findings demonstrate that SARS-CoV-2 infection initiates tissue-wide antiviral signaling in the OE that persistently disrupts OSN function, providing a mechanistic framework for COVID-19-associated anosmia.

neuroscience↗

bioRxiv2026

Uhrf1 loss disrupts Ctcf-associated chromatin organization during early mouse embryogenesis

UHRF1 is a chromatin-binding protein essential for maintaining DNA methylation and histone modification states, yet its integrated role in vivo remains incompletely understood. To define its function, we generated conditional Uhrf1 knockout embryonic stem cells (ESCs) and embryos. Uhrf1-/- ESCs exhibited near-complete loss of 5mC and 5hmC but maintained pluripotency, whereas Uhrf1-null embryos developed normally until E8.5 and then failed to develop further by E9.5, phenocopying Dnmt1 loss. Single-cell multi-omic (ME-seq) profiling of E8.5 embryos revealed impaired lineage stabilization, widespread hypomethylation, and disrupted chromatin architecture. Uhrf1 loss was associated with altered CTCF-associated chromatin signal, broad remodeling of chromatin contacts, altered cis-regulatory relationships, and reduced predicted BMP-related ligand-receptor communication, particularly within neural crest populations. These findings identify Uhrf1 as a central regulator that tightly couples DNA methylation maintenance to 3D genome organization during gastrulation, thereby, directing early lineage specification and positioning Uhrf1 as a pivotal mediator of epigenetic information transfer during early embryogenesis. HighlightsO_LIUhrf1 knockout ESCs show global loss of 5mC/5hmC but maintain pluripotency. C_LIO_LIUhrf1-null embryos develop normally until E8.5 but die by E9.5 with severe defects. C_LIO_LISingle-cell multi-omics revealed disrupted chromatin, transcription, and lineage stability upon knockout. C_LIO_LILoss of Uhrf1 alters CTCF-associated chromatin signal, predicted BMP-related communication, and cis-regulatory relationships. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738670v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@15bf31dorg.highwire.dtl.DTLVardef@1b601ecorg.highwire.dtl.DTLVardef@386018org.highwire.dtl.DTLVardef@12ce99f_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2026

Rapid Intracellular Delivery of Human Heat Shock Protein 72 Prevents Memory Loss and Inhibits Neurodegeneration up to 9 Days After a Blast Injury

Traumatic brain injuries (TBIs) are increasingly prevalent among military service members and are associated with long-term neurological impairment and neurodegeneration. Heat shock protein 72 (HSP72) has demonstrated cytoprotective properties and has been shown to cross the blood brain barrier in rat models of blast injury, remaining in brain tissue for up to 12 hours. In this study, we evaluate engineered Fv-HSP72 variants for their ability to reduce neurodegeneration and preserve short-term memory following blast-induced TBI. Male Sprague-Dawley rats were assigned to 9 groups of n = 8 rats. Animals were either not exposed to blast (Sham), exposed to blast (Blast Only), blast exposed and given buffer (Vehicle), or blast exposed and treated with one of three Fv-HSP72 variants, dosed at 10 or 30mg/kg at 15m post-blast. Blast exposure was generated using an Advanced Blast Simulator (ABS) producing positive static pressure to model moderate to severe blast injury. Animals were euthanized 48 hours post injury for neurodegeneration and immunologic biomarker analysis. After selecting an effective Fv-HSP72 variant using the biomarker data, additional rats were divided into Sham, Vehicle, and Fv-HSP72 treatment groups to evaluate short-term memory function through the Novel Object Recognition (NOR) test on days 2 and 8 post-blast. Analysis of cortical and spinal cord tissues demonstrated a statistically significant reduction in expression of neurodegenerative markers of Tau phosphorylation and glial injury (GFAP) for rats receiving a single dose of our clinical candidate, RBB012-CTB. In fact, the drug drove astrogliosis toward a neuroprotective state in blast exposed rats. In the NOR assay, Fv-HSP72 treated rats showed improved recognition performance, indicating preservation of short-term memory function. With similar biomarker results obtained for a controlled cortical impact injury model published elsewhere (Chan et al. manuscript submitted), the analyses suggest Fv-HSP72 is neuroprotective following a blast injury as well. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly preventing learning and memory loss in rats for up to 9 days after a blast injury.

neuroscience↗

bioRxiv2026

Machine learning cross-platform proteomic imputation enables protein quality scoring and replication of epidemiological associations

High-throughput affinity-based proteomics has advanced biomedical research, yet fundamental, persistent discordance between mainstream platforms (SomaScan and Olink) routinely undermines the replication of findings. This platform-driven non-replication complicates downstream biological validation and biomarker prioritization. Here, we develop a machine learning-based framework for cross-platform protein value imputation to resolve this translational bottleneck. Using paired proteomic data measured by both SomaScan and Olink from 5,325 participants of the Multi-Ethnic Study of Atherosclerosis, we developed models to impute cross-platform measurements and applied them to two independent and demographically distinct cohorts (Cardiovascular Health Study [N=3,171] and UK Biobank [UKB; N=41,405]) for external validation. Our bi-directional model 1) established an imputation performance-based protein fidelity index, validated against gold-standard measurements from Atherosclerosis Risk in Communities study (N=101) and Nurses Health Study (N=54), 2) enabled imputation of platform-exclusive protein measurements, and 3) facilitated calibration of overlapping proteins. We demonstrate the utility of this framework through three applications: 1) fidelity-informed analyses enhanced the replication of biomarker discovery, 2) recovery of SomaScan signals that were previously inaccessible in UKBs original Olink measurements, and 3) improved replication performance for overlapping proteins. Our study offers a translational roadmap that allows researchers to achieve reliable epidemiological replication, target specific assays for future optimization, and prioritize biological signal over platform noise.

bioinformatics↗

bioRxiv2026

Rapid Intracellular Delivery of Human Heat Shock Protein 72 Inhibits Neurodegeneration and Oxidative Damage After a Traumatic Brain Injury

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

neuroscience↗

bioRxiv2026

Resolving heterogeneity of targeted lipid nanoparticles through solution-based biophysical analyses

Targeted lipid nanoparticles (tLNPs) represent the next frontier in nucleic acid therapeutics, enabling cell-specific delivery through covalent attachment of targeting ligands that drive receptor-mediated uptake. tLNPs are particularly promising for pregnancy-associated applications where precise on-target delivery is required to minimize maternal toxicity and protect fetal health. Yet, their rational design is limited by an incomplete understanding of how tLNP physicochemical properties influence biological performance. Conventional LNPs already exhibit pronounced heterogeneity in size, composition, and RNA loading, which is further amplified in tLNPs by variability in ligand attachment and surface density. Because traditional analytical methods report only ensemble-averaged properties, the nanoscale diversity of tLNPs remains unresolved. Here, we find that tLNP functional behavior is governed by previously inaccessible, structurally distinct tLNP subpopulations that are not captured by bulk measurements. We utilize asymmetric flow field-flow fractionation integrated with in-line UV spectral analysis, light scattering, and synchrotron small-angle X-ray scattering (AF4-UV-DLS-MALS-SAXS) to resolve ligand-dependent tLNP subpopulations that differ in size, shape, composition, and relative abundance. We find that protein conjugation preserves the internal lipid-RNA nanostructure of base LNPs but substantially increases particle heterogeneity, particularly for larger and multivalent targeting ligands. Despite increased heterogeneity, tLNPs functionalized with higher-avidity ligands achieve more effective targeted placental RNA delivery in mice, suggesting that binding avidity can offset the functional consequences of polydispersity. Chemometric SAXS analyses reveal that only SAXS-resolved tLNP subpopulations, not ensemble-averaged parameters, correlate with targeted placental transfection in vivo, whereas bulk-derived physicochemical metrics more strongly associate with nonspecific hepatic delivery. Together, this work harnesses a separation-coupled biophysical platform to resolve previously inaccessible tLNP subpopulations and demonstrates that subpopulation nanoscale structure, rather than bulk-averaged properties, dictates targeted RNA delivery. These insights provide a mechanistic foundation for rational engineering of next-generation precision targeted RNA LNP therapeutics.

bioengineering↗

bioRxiv2026

Simultaneous broad protection against Ebola Sudan, Marburg and Lassa viruses conferred by a DNA primed MVA-vectored multivalent vaccine

Sub-Saharan Africa continues to experience recurrent outbreaks of zoonotic viral diseases that spill over unpredictably from animal reservoirs into human populations. In many regions, mpox co-circulates with viral hemorrhagic fevers (VHFs) caused by Ebola Sudan virus (SUDV), Marburg virus (MARV), and Lassa fever virus (LASV). Overlapping clinical syndromes that these VHF cause challenge surveillance, diagnostics and timely deployment of effective countermeasures. A single vaccine capable of protecting against these biologically and genetically distinct pathogens would markedly reduce the cost and complexity of outbreak response, lessen dependence on emergency international aid, and strengthen long-term health system resilience. Here, we report on the development of an MVA-based mpox vaccine engineered to express computationally designed, broad-coverage antigens targeting SUDV, MARV and LASV. In preclinical challenge studies, this multivalent vaccine elicited robust immune responses and conferred significant protection against lethal infection from all three pathogens in parallel challenge experiments. These findings establish preclinical proof-of-concept for a single, broadly protective VHF vaccine and support its clinical development for deployment across diverse settings in Sub-Saharan Africa. SignificanceOutbreak control in Sub-Saharan Africa is challenged by the co-circulation of different high consequence human infections such as mpox and diverse viral hemorrhagic fevers (VHF) such as SUDV, MARV, and LASV pathogens. These VHFs have overlapping early clinical syndromes, complicating triage and delaying effective targeted interventions. We developed a single MVA-based vaccine encoding computationally designed, conserved antigens from all three VHFs encoded within the MVA vector analogous to the licensed mpox vaccine. In simultaneous challenge models, this multivalent vaccine elicited robust humoral and cellular responses and conferred significant protection against lethal infection by each hemorrhagic fever pathogen. This work provides preclinical proof-of-concept for a unified, broadly protective countermeasure compatible with existing MVA-mpox vaccine manufacturing and deployment experience. By reducing dependence on rapid differential diagnostics and streamlining logistics relative to maintaining multiple pathogen-specific vaccine stockpiles, this approach can lower costs, accelerate response, and increase equity of access during syndromic outbreaks. The platforms engineered antigen breadth and human safety profile of MVA together support a pragmatic translational pathway toward clinical evaluation and regional readiness for zoonotic spillover events that are intensifying with human and environmental changes.

immunology↗

bioRxiv2026

Spatiotemporal 4D Whole-cell Modeling of a Minimal Autotroph Reveals Central Carbon Metabolism Regulated Locally by Protein Megacomplexes via Post-translational Modifications under Light Disturbance

Photosynthetic microorganisms rely on multiple pathways in central carbon metabolism to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes spanning disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. To address this complexity beyond genome-based inference and traditional modeling, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial organization of enzymatic and molecular processes in central carbon metabolism under light perturbation. We employ a perturbation-based research design to experimentally generate time-series, multi-omics measurements that provide molecular descriptors and cryo-ET images as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulating the structural assembly of a protein megacomplex, the "dark complex," modulate metabolic flux at a conserved regulatory node of the Calvin-Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates that post-translational regulation not only buffers transcriptional noise and diffusion-driven fluctuations but also stabilizes phenotypic outcomes, underscoring the importance of spatial heterogeneity in phenotypic robustness. This ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and central carbon metabolism addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4. Significance StatementThis work advances 4D whole-cell modeling by presenting the first spatiotemporal simulation of a photosynthetic autotroph using the Lattice Microbes platform. Using Prochlorococcus marinus MED4, we show that subcellular spatial organization of organelles, diffusion constraints, and redox regulation collectively shape central carbon metabolism across orders of magnitude in space and time. Through a perturbation-based strategy that generates multi-omics data sets over time, we construct and validate a spatially and temporally resolved model of MED4, constrained by high-resolution (10 nm) cryo-electron tomography. Our results highlight the importance of localized biochemical reactions and redox-dependent post-translational modification of enzymes in regulating carbon fixation in a noisy environment under light disturbance. This study establishes a spatiotemporal, whole-cell physiology modeling framework as a transformative tool for uncovering multiscale regulatory responses to environmental gradients.

systems biology↗

bioRxiv2025

Consensus Co-Expression Analysis Identifies A Common Set Of Co-Expressed Genes Associated With Diabetic Peripheral Neuropathy And Chemotherapy-Induced Peripheral Neuropathy

BackgroundDiabetic peripheral neuropathy (DPN) and chemotherapy-induced peripheral neuropathy (CIPN) are major clinical challenges with limited therapeutic options. While these conditions arise from different causes, they may share common molecular mechanisms that could be targeted for intervention. MethodsWe performed consensus weighted gene co-expression network analysis (WGCNA) on two publicly available datasets: GSE185011 (DPN vs. healthy controls in peripheral blood mononuclear cells) and GSE173610 (paclitaxel-treated vs. control iPSC-derived sensory neurons). After filtering all but the most variable genes, consensus analysis was used to identify conserved co-expression modules across both conditions. ResultsConsensus analysis identified a 193-gene module (ME3/brown) significantly associated with both DPN (correlation=0.817, p=0.0040) and CIPN (correlation=0.971, p=0.0060). Functional enrichment analysis of this module revealed pathways related to Glycolysis, FoxO signaling, Apoptosis, and Autophagy. ConclusionsOur analysis reveals a convergent molecular signature underlying both DPN and CIPN, centered on metabolic reprogramming, transcriptional stress, and programmed cell death. These findings provide a systems-level framework for developing therapies targeting shared pathological mechanisms.

systems biology↗

bioRxiv2025

Bruch's Membrane Contributes to the Structural Integrity of the Human Eye

PurposeTo investigate the contribution of the Bruchs membrane and sclera tissues to the overall structural integrity of the ocular wall. MethodsTwenty-three human globes were subjected to biomechanical testing. A piece of sclera measuring 5 x 5 mm was carefully removed at the nasal region, 2 mm away from the optic nerve head. The intraocular pressure was increased at approximately 1 mmHg/s until Bruchs membrane-uvea-retina-tissue layer (BMUR) ruptured. Next, strips of sclera and Bruchs membrane-choriocapillaris (BMC) complex were isolated from the superior fundus region. Uniaxial tension tests were performed at a strain rate of 0.01/s and sampling rate of 15 Hz. The tangent moduli of the BMC and sclera at 0.01, 0.02 and 0.03 strains were compared. ResultsThe rupture pressure of the BMUR was 98.1 {+/-} 21.4 mmHg. The tangent moduli of the BMC at 0.01, 0.02 and 0.03 strains were 2.96 {+/-} 1.44 MPa, 7.68 {+/-} 1.78 MPa and 9.43 {+/-} 2.11 MPa, respectively, and the tangent moduli of the sclera at 0.01, 0.02 and 0.03 strains were 1.09 {+/-} 0.80 MPa, 2.72 {+/-} 1.67 MPa and 5.69 {+/-} 3.27 MPa, respectively. ConclusionThe BMUR was able to sustain relatively high IOP before rupturing. The uniaxial tensile tests showed that the BMC tangent moduli were about 3 times of those of the sclera at strains of 0.01 and 0.02. Although the sclera is approximately 47 times thicker, the BMC is still likely to make a significant contribution (3.51% to 7.42% at strain <0.03) to the overall structural strength of the ocular wall.

bioengineering↗

bioRxiv2025

Electrophysiological Brain Connectivity and Subjective States Evoked by Electrical Stimulation of the Human Mediodorsal Thalamus

Recent advances in human intracranial EEG (iEEG) have enabled new investigations into the role of the thalamus in human brain functions. In this study, we applied direct intracranial electrical stimulation (iES) to the mediodorsal (MD) subregion of the thalamus using both high-frequency (50 Hz, iESHF) and low-frequency (0.5 Hz, iESLF) procedures to examine its impact on conscious experience and causal brain connectivity in 30 patients with focal refractory epilepsy (128 electrode contacts; 4 {+/-} 1 MD sites per patient). iESHF of the MD elicited reportable changes in conscious experience in 11 of 12 patients (39 sites; 83 stimulations across 27 unique pairs) - predominantly in the visceral, emotional, or somatosensory domains and often described as unpleasant without any lateralization effect. Our connectivity analyses based on iESLF revealed that the cingulate and insular cortices produced stronger electrophysiological responses in the MD (inflow connectivity) than did the sites in the prefrontal cortex (PFC) within the same individuals. Moreover, MD stimulation showed its strongest outflow connectivity to the cingulate, insular, and PFC regions, all significantly stronger than to medial temporal lobe (MTL) structures. Notably, inflow from both MTL and insula sites to the MD were significantly stronger than their reverse directions, indicating clear asymmetry in connectivity. These findings provide direct evidence that stimulation of the human thalamus can modulate conscious experience. They also highlight the extensive bidirectional connectivity between the MD and cingulate and insular cortices along with asymmetric connectivity between the MD and MTL and insula sites in the human brain. SIGNIFICANCE STATEMENTOur findings provide a functional and causal map of the mediodorsal thalamus (MD) in the human brain. We provide direct evidence that stimulation of the human thalamus can modulate conscious experience. This study also holds clinical and translational value for identifying thalamic pathways involved in the propagation and generalization of seizures, especially seizures involving the medial temporal lobe, as well as for neuromodulation in epilepsy and other neuropsychiatric disorders, as MD stimulation may not be well-tolerated in human subjects.

neuroscience↗

bioRxiv2025

Temporal Dynamics of Antigen-Specific T Cell Expansion in Primary SARS-CoV-2 Infection

Quantifying T cell response during primary infection in humans is crucial for understanding adaptive immunity. Leveraging a controlled human challenge to SARS-CoV-2, we characterized antigen-specific T cell response within and across individuals. Notably, individual clones reached similar maximum frequencies despite differences in the timing of their peak expansion. Mathematical modeling showed that this observation is consistent with precursor frequency, but not TCR signal strength, as the source of inter-clonal variability. Single-cell profiling revealed distinct temporal programs for CD4+ and CD8+ T cells, with CD4+ cells expanding earlier but contracting to a lower frequency. Clones with similar receptors, likely recognizing the same antigen, expanded at similar times. Together, these findings highlight how clone-intrinsic properties such as precursor frequency and lineage shape T cell clonal kinetics. These insights provide a quantitative framework for understanding T cell response in humans, with implications for vaccine design.

biophysics↗

bioRxiv2025

Immunogenicity and Efficacy of Digitally Immune Optimised H1N1 Vaccine Candidates in Swine and Murine Animal Models

Influenza A virus (IAV) zoonotic transmission and constant evolution in multiple species heightens the risk of emerging novel strains at the human-animal interface. Composite antigens including hemagglutinin (HA), neuraminidase (NA), and matrix-2 (M2) proteins were computationally designed to maximize the breadth of the immune response elicited to human seasonal, pandemic, and zoonotic H1N1 IAVs. Mouse hyperimmune serum raised against these antigens demonstrated broad H1 neutralization and N1 inhibition activity. To enhance immunogenicity, the antigens were combined as a single DNA expression construct (DVX-H1N1). Studies in the well-recognized swine model for human influenza demonstrated that DVX-H1N1 immunization induced broad, neutralizing antibody responses and markedly reduced nasal shedding of viral RNA following challenge with 1A.3.3.2 subclade strain A/swine/England/1353/2009 (H1N1). An effective immune response and reduction in virus shedding was observed in pigs immunized with a whole inactivated virus (WIV) vaccine homologous to the challenge strain but not with a human-origin seasonal WIV vaccine. Overall, we demonstrated broad immunogenicity and efficacy of the DVX-H1N1 vaccine candidate, benchmarked against relevant IAV H1N1 strains in vitro and in vivo in mice and pigs. IMPORTANCEThe zoonotic potential of swine-origin IAVs is a recognized global health threat. Vaccination remains the most effective intervention against influenza; protecting at the population level by preventing nasal shedding and transmission, but also in individuals by limiting clinical disease, particularly by reducing the severity of lung infection. The World Health Organization (WHO) spearheads biannual surveillance efforts to review evolving virus strains and vaccine antigens at Vaccine Candidate Meetings (VCM) to recommend strain updates for the human seasonal influenza vaccine and for pandemic preparedness purposes. However, the strain selection approach is complex and efficaciousness of seasonal influenza vaccines still varies significantly based on the accurate matching of the predicted strains in circulation with the manufactured vaccine antigens. This emphasizes the need for next-generation influenza vaccines that improve the breadth and longevity of immunity. We describe a computationally optimized DNA vaccine with broad immunogenicity and robust efficacy in the pig model.

immunology↗

bioRxiv2025

Enhanced Medium-Chain Fatty Acid Production in Megasphaera elsdenii via Adaptive Laboratory Evolution and Genomic Insights

The shift toward a sustainable bioeconomy has increased interest in microbial platforms capable of producing valuable fatty acids. Megasphaera elsdenii is emerging as a promising candidate for fatty acid bioproduction, although its performance varies depending on the strain. This study compares two M. elsdenii strains, JCM 1772 (ATCC 25940) and JCM 35779, revealing that JCM 35779 produces up to 5.8 times more hexanoic acid than JCM 1772. Comparative genomic analysis identified key differences between the strains, including the loss of enzymes involved in the pyruvate-to-butyric acid pathway in JCM 35779, indicating a rerouting of carbon flux that favors chain elongation toward hexanoic acid instead of butyric acid. Overall, this work links genomic variation to metabolic phenotype, establishes JCM 35779 as a superior medium-chain fatty acid producer, and supports the development of M. elsdenii as an effective platform for sustainable production of medium-chain fatty acids. HighlightsO_LIPig-feces M. elsdenii shows >5 times higher HA production than its rumen strain. C_LIO_LIGenomic analysis reveals strain-specific deletions in pathways for MCFA synthesis. C_LIO_LIThis includes etfB2, cat1 and abfD. C_LI

microbiology↗

bioRxiv2025

IL-6 is a Key Factor in the Formation of Gut Tissue Resident Memory T Cells from Naïve T cells

Tissue resident memory CD4+ T cells (TRMs) populate mucosal sites and play a critical role in local immune responses. Gut TRM cells persist for extended periods in the gut mucosa where they rapidly respond to invading pathogens and provide long lasting protection. This study investigates the factors that mediate differentiation of naive CD4+ T cells into cells presenting a gut TRM phenotype. Naive CD4+ T cells were cultured under conditions that mimicked mucosal environments. This included signaling through MAdCAM-1 in the presence of Retinoic Acid (RA) and TGF-{beta}. This combination of stimuli primed naive CD4+ T cells to adopt a TRM phenotype. However, to fully differentiate into TRMs an additional soluble factor provided by memory T cells was required. Our results identified IL-6 as a key factor that induces the expression of TRM -associated markers, including CD69, CD103 and CCR5. This unique combination of stimuli promoted TRM differentiation despite low level proliferation. TRM differentiation was mediated through JAK/STAT signaling, and antagonists that target JAK/STAT pathways suppressed MAdCAM-1 mediated TRM cell formation. Our findings revealed that MAdCAM-1 works together with TGF-{beta}, RA and IL-6 in this process. Such information may aid in the design of next generation adjuvants and effective mucosal vaccines. Additionally, each of these factors may be targeted to treat excessive gut inflammation associated with conditions like inflammatory bowel disease. Overall, these findings provide new strategies aimed at modulating immune responses to invading pathogens and identify therapeutic approaches toward regulating gut inflammation. Author SummaryImmunologists and vaccinologists have long been interested in strategies aimed at boosting immune responses in mucosal tissues where pathogens are first encountered. Gut Tissue resident memory T cells (TRMs) play a central role in gut homeostasis and immunity. They provide a rapid long-lasting protection against pathogens. TRMs must target harmful pathogens and at the same time tolerate harmless commensal bacteria. This balance between immunity and tolerance in the complex environment of gut tissues is essential to the maintenance of gut homeostasis. In this study we have identified key factors present in the gut tissue milieu that are involved in TRM differentiation from naive T cells. These findings point to new therapeutic approaches that can potentially target immune responses in gut tissues and may help in developing effective mucosal vaccines. Conversely, these factors may be targeted in excessive gut inflammation involved in conditions like inflammatory bowel disease (IBD). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/660664v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@14e6015org.highwire.dtl.DTLVardef@860b93org.highwire.dtl.DTLVardef@61f8c8org.highwire.dtl.DTLVardef@142b48_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Box C/D snoRNPs and MDT-15/MED15 regulate mitochondrial surveillance via fatty acid metabolism

Text AbstractIn response to constant homeostatic threats, organisms have developed complex regulatory networks to monitor cellular functions and restore normal function. Here, we identify MDT-15 and its effectors, the fatty acid desaturases FAT-5, FAT-6, and FAT-7, as activators of the Ethanol and Stress Response (ESRE) mitochondrial surveillance pathway. Our data show that box C/D snoRNPs, which were previously linked to ESRE activation, also regulate FAT-6 and FAT-7 protein levels. Notably, knockdown of mdt-15 or fib-1, a component of box C/D snoRNP complex, increased accumulation of the mitophagic activator PINK-1, the first step in licensing mitophagy, suggesting a relationship between ESRE surveillance and mitophagic activation. Supplementation with downstream unsaturated fatty acid products of FAT-6 and FAT-7 enhanced ESRE and mitophagic activation, but did not affect UPRmt. Since fatty acids activated ESRE and PINK-1 in wild-type and mutant genetic backgrounds, they are likely to act via a mechanism independent of FAT-6 and FAT-7 function. Our results provide insight into a novel interplay between box C/D snoRNPs, MDT-15, and fatty acids in the regulation of mitochondrial surveillance and mitophagy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/656193v2_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@92f749org.highwire.dtl.DTLVardef@a8f496org.highwire.dtl.DTLVardef@51b28dorg.highwire.dtl.DTLVardef@1a15374_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Plasmodium falciparum DNA repair dynamics reveal unique roles for TLS polymerases and PfRad51 in genome diversification

The haploid malaria parasite, Plasmodium falciparum, evolved a unique cohort of DNA repair pathways enabling the parasite to survive in a vertebrate host red blood cell and the mosquito vector. P. falciparum chromosomes are partitioned into a highly conserved core genome and remarkably diverse, largely subtelomeric regions that contain genes encoding important parasite virulence factors. The molecular mechanisms that maintain this chromosomal structure have not been identified. Here, we describe specific DNA repair pathways that differentiate between hypervariable subtelomeric and conserved core regions of the genome. Based on our previous work, we hypothesized that there are potentially important interactions between translesion (TLS) and homologous recombination (HR) pathways for the diversification of multicopy gene families in P. falciparum. Thus, we created knockout parasite lines of the DNA repair enzymes: PfRad51 and the TLS polymerases PfPol{zeta} and PfRev1. We identified that irradiation hypersensitivity varied across the cell cycle for TLS{Delta} parasites and was uniform across the erythrocytic cycle for PfRad51{Delta} parasites, highlighting the variable roles of these pathways. However, important interactions between these pathways were found when we studied directed double strand break (DSB) repair, which revealed a difference in the DNA damage response according to chromosomal location. PfRad51 was essential for HR-mediated repair in the core genome. In contrast, we identified a Rad51 independent homology-directed repair in all three of our knockout lines when a DSB was made in the subtelomeric region of the chromosome. We propose that this differential DNA damage response maintains the distinction in diversification across the chromosome.

microbiology↗

bioRxiv2025

Shear stress targeted delivery of nitroglycerin to brain collaterals improves ischaemic stroke outcome

In patients with ischaemic stroke, retrograde perfusion of the penumbra by the leptomeningeal collateral vessels (LMCs) is a strong predictor of clinical outcome, thus raising the possibility that enhancing LMC flow could offer a novel therapeutic approach. Here, using computational modelling we show that LMCs experience elevated fluid shear stress that is significantly higher than that in other blood vessels during ischaemic stroke in animals and humans. We take advantage of this to selectively enhance flow in LMCs using shear-activated nanoparticle aggregates carrying the vasodilator nitroglycerin (NG-NPAs) that specifically release drug in regions of vessels with high shear stress ([≥]100 dyne/cm2). The NG-NPAs significantly increased LMC-mediated penumbral perfusion, decreased infarct volume, and reduced neurological deficit without altering systemic blood pressure in a rat ischaemic stroke model. The NG-NPAs also did not cause known common side effects of systemic nitrate administration, such as systemic hypotension, cerebral vascular steal, cortical vein dilation, or intracranial pressure elevation. Systemic administration of free NG at the maximal tolerated dose, which was ten times higher than the dose of NG used in the NG-NPAs, did not enhance LMC perfusion and dropped blood pressure. Thus, packaging NG within shear-activated NPAs can potentially enable this widely available vasodilator to become a highly effective therapeutic for ischaemic stroke.

physiology↗