Biology subjects

Joshi, A.

Publications and source records attributed to Joshi, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Divergent genotype-phenotype maps determine the capacity for adaptation to antibiotics among Enterobacteria

What dictates the extent to which a bacterial strain can evolve under antibiotic pressure remains poorly defined, despite its implications for the effectiveness of antibiotics in clinical practice. In this study, we compare the evolution of Escherichia coli and Klebsiella aerogenes, two medically relevant members of Enterobacteriaceae, under colistin and trimethoprim selection. We discover that the mere presence of effector genes of resistance, as well as conserved gene regulatory architecture, do not necessarily translate to similar evolvabilities during antibiotic challenge. Instead genotype-phenotype maps of adaptive mutations, which can diverge at different biological levels, govern how effectively a strain evolves resistance. Both, E. coli and K. aerogenes, demonstrated strikingly parallel capacities for adaptation to trimethoprim, as a result of conserved mechanisms of resistance, including drug target modification and efflux. On the other hand, E. coli was driven to extinction by colistin, while K. aerogenes was able to evolve a high level of colistin resistance. Like the better studied K. pneumoniae, K. aerogenes acquired mutations in the BasSR (also called PmrAB) and PhoQP two-component signalling pathways under colistin selection. These mutations increased the expression of chromosomally-encoded lipopolysaccharide (LPS) modifiers, EptA and Arn. Similar mutations in E. coli were unable to confer protection against colistin, despite successfully up-regulating homologous effectors genes. However, expression of MCR-1, a horizontally acquired EptA-homolog found in clinical strains, conferred high level colistin resistance to E. coli. This difference in the intrinsic capacity to adapt to colistin was reflected in frequencies of resistance among clinical isolates from around the world. It also altered the dependency of Escherichia and Klebsiella species on horizontal gene acquisition for colistin resistance. Our study highlights the importance of understanding pathogen-specific genotype-phenotype maps of drug resistance to better predict long-term effectiveness of antibiotic therapies and define resistance-proof treatment strategies.

microbiology↗

bioRxiv2026

Deciphering efficient metabolic pathways in an anaerobic gut fungus coupled with a syntrophic methanogen

Anaerobic gut fungi (AGF) are an integral part of the herbivore gut microbiome, primary decomposers of lignocellulose, and play a crucial role in establishing foundational links of syntrophic associations. The syntrophy with methanogens appears beneficial for mitigating feedback inhibition and results in the augmentation of AGF activity. However, the thermodynamic advantages of the presence of methanogens to AGF remain poorly understood. This study explores the preferred pathways adopted by Cyllamyces sp. and Methanobrevibacter olleyae, using 13C-glucose as a substrate and spiked with malate and pyruvate in different experimental set-ups. The results indicated reduced biomass formation by Cyllamyces sp. in monoxenic culture with Methanobrevibacter olleyae compared to axenic AGF culture supplemented with glucose. Additionally, metabolic analyses revealed the pyruvate:ferredoxin oxidoreductase (PFO) as a key contributor to pyruvate metabolism. Malate supplementation notably enhanced the growth of Cyllamyces sp. and M. olleyae, whereas pyruvate supplementation enhanced the growth of Cyllamyces sp. only in axenic culture. This investigation provides insights into the preferred metabolic pathways operating in the hydrogenosomes of axenic anaerobic gut fungi (AGF) cultures and monoxenic AGF-methanogen co-cultures.

microbiology↗

bioRxiv2026

Alcohol Use Disorder and Smoking-Associated Molecular Alterations in Human Prefrontal Cortex in Single Nucleus (sn) RNA-Seq

Chronic smoking worsens alcohol related brain injury and impairs neurocognitive recovery, yet the cell type specific molecular interactions between alcohol use disorder (AUD) and smoking in the human brain remain largely unexplored. We analyzed single-nucleus RNA-seq from the prefrontal cortex of 73 individuals (614,932 nuclei, 24 cell types), comparing AUD versus controls, smoking versus controls, and combined AUD+smoking versus controls. In excitatory neurons, neuroinflammatory, complement, GPCR, kinase, proteostatic, and extracellular-matrix pathways confined to one or two subtypes in either condition alone expanded across nearly all subtypes when AUD and smoking co-occurred. Inhibitory neurons showed a parallel but distinct expansion, additionally recruiting translational regulation, RNA splicing, and deubiquitination pathways. Smoking alone produced widespread repression of bioenergetic, proteostatic, and synaptic pathways in excitatory neurons, whereas AUD produced mixed pathway activation and repression in excitatory neurons and broad inflammatory activation in inhibitory neurons. In the combined condition, microglia exhibited paradoxical repression of phagocytosis, TYROBP signaling, and translational machinery, consistent with an exhaustion like state, accompanied by an apparently compensatory shift in inflammatory signaling toward astrocytes, oligodendrocyte precursor cells, and oligodendrocytes, which showed coordinated inflammatory activation. Vascular cells showed an endothelial-VLMC dichotomy, with endothelial repression of RNA processing and proteostasis contrasting with perivascular activation. Our analysis identifies bioenergetic and proteostatic stress as the predominant signature of smoking, whereas immune and metabolic activation characterize AUD. In AUD+Smoking, these alterations extend across more neuronal subtypes and include neuroinflammation, complement activation, altered signaling, proteostatic stress, and post-transcriptional remodeling associated with greater cortical dysfunction in co-occurring AUD and smoking.

genomics↗

bioRxiv2026

Revisiting the evolution of population stability due to selection for rapid development and early reproduction in Drosophila: the role of generation length

The ubiquity of stable populations in nature generated considerable interest in how population stability might evolve, especially after the realization that higher per capita population growth rates typically yield unstable dynamics. Yet, most empirical and theoretical treatments implicitly assume that population dynamics and stability are invariant to generation length, an assumption that remains largely untested. Theory suggested that population stability could evolve as a correlated response to life-history evolution, which was first experimentally demonstrated using D. melanogaster populations selected for rapid development and early reproduction (FEJs). Constancy stability of FEJs evolved to be higher than their ancestral controls (JBs), likely due to correlated reductions in fecundity and pre-adult survivorship. However, that study assessed stability on a 21-day generation length (matching the JBs), resulting in a considerable mismatch with the generation length of FEJs (10 days). This raises a broader question of whether the observed differences in stability reflect evolved life-history changes or are an artifact of the generation length at which the populations were assayed. To address this, we assessed the stability of FEJs, JBs, and relaxed-selection populations derived from the FEJs (CRFs and FRFs) across two generation lengths (12 and 18 days), tracking 320 small populations for 27 generations. Contrary to the earlier findings based on a 21-day generation length study, FEJs did not differ in constancy from JBs when assayed at a shorter generation length. Thus, even a modest difference in life-cycle length can significantly influence population stability, thereby underscoring the need to account for generation length when comparing stability across populations. Interestingly, constancy and persistence stability of selection regimes evolved in opposite directions, highlighting the need to assess stability along multiple axes. We discuss these results using an empirical framework, emphasizing its utility over simple population growth models for a richer understanding of population dynamics and stability.

ecology↗

bioRxiv2026

Modeling steady state thermoregulation of near-term human fetus

Maternal thermal strain is associated with adverse pregnancy outcomes, yet fetal temperatures cannot currently be directly measured, limiting quantification of fetal thermal strain. Here, we develop two steady-state models for estimating internal temperatures in a near-term fetus. First, we improve the only previously published human fetal thermoregulation model, deriving a closed-form solution within its simplified uniform-cylinder representation. Second, we introduce a multilayer, anatomically segmented model that resolves tissue-specific temperatures. Both couple the fetal body to central blood pool and amniotic fluid compartments and incorporate a new placenta-umbilical cord heat-exchanger representation. Predictions agree with available intrauterine scalp measurements, with fetal core and head-center temperatures approximately 0.5{degrees}C and 0.8{degrees}C above maternal core, respectively. Physiologically plausible changes in umbilical cord heat-exchanger effectiveness or blood flow increased fetal temperatures by approximately 0.3{degrees}C. These models enable estimation of otherwise inaccessible temperatures, while the multilayer formulation lays a foundation for transient, coupled maternal-fetal thermoregulation modeling.

biophysics↗

bioRxiv2026

Listening with your heart: The heartbeat shapes auditory object formation by suppressing the early neural response to sound

Interoceptive sensations, arising from the body's visceral organs such as the heart, are known to impact exteroceptive sensory perception. The classical explanation for heart-to-brain influence, the Baroreceptor Hypothesis, posits that baroreceptors firing during the systolic blood pressure peaks that follow each heartbeat suppress the magnitude of neural responses to exteroceptive sensations. More recent work, however, has demonstrated qualitative (rather than merely magnitude) differences in perception as a function of the cardiac cycle; since it is not obvious how the Baroreceptor Hypothesis could explain these findings, even in principle, they have often been characterized as incompatible. We propose baroreceptor-related suppression of early sensorineural responses need not manifest simply as suppression of corresponding conscious percepts. In a validated computational model of auditory cortex that segregates an ambiguous tone sequence into either one or two auditory objects or "streams," we found suppression of the neural response to one tone type increases the likelihood that tone is parsed into a distinct stream. We subsequently verified this prediction empirically: when presenting such ambiguous sequences to human participants in a manner such that one tone type only occurs during cardiac systole, the initial neural response to that tone -- indexed by the electroencephalographic (EEG) frequency-following response (FFR) -- is indeed suppressed, while participants report hearing the sequence as two separate sounds streams more frequently. Thus, suppression of early sensorineural responses can be sufficient to explain qualitative, not just magnitude, differences in perception when considered in the context of larger neural circuits.

neuroscience↗

bioRxiv2026

Bioactive Spermidine-Crosslinked DNA Hydrogel for rapid homeostasis and accelerated wound healing

Damage to the skin by trauma, burns, or surgical procedures often results in uncontrolled bleeding, which remains a leading cause of preventable death following injury, yet most conventional hemostatic materials are engineered solely to arrest bleeding and often adhere strongly to the wound bed, causing pain, rebleeding, and disruption of newly formed tissue upon removal. Here, we report a DNA hydrogel that structurally mimics neutrophil extracellular traps (NETs) and is crosslinked using a bioactive small molecule with potent autophagy-inducing, cardioprotective, anti-inflammatory, antioxidant, and mitochondria-protective properties, integrating rapid hemostasis with active support for tissue regeneration in a single biomaterial. The DNA network provides an intrinsically biocompatible, biodegradable scaffold capable of recruiting platelets and erythrocytes to achieve rapid clot formation, while the bioactive crosslinker is released as the network degrades, delivering a sustained cytoprotective and anti-inflammatory stimulus directly at the wound site. The hydrogel was characterised physiochemically and evaluated for cytocompatibility, hemolytic potential, hemostatic efficacy, and wound-healing performance in a murine model. Results demonstrate that the bioactive-crosslinked DNA hydrogel achieves rapid, effective hemostasis, while accelerating wound closure and supporting regenerative tissue remodelling. This dual-function platform offers a promising strategy for next-generation wound-care biomaterials that unite immediate bleeding control with accelerated, natural tissue healing.

bioengineering↗

bioRxiv2026

Tumor-hepatocyte crosstalk drives a hepatic lactate-TGF-β axis of CD8⁺ T cell exhaustion and immunotherapy resistance in small-cell lung cancer liver metastases

PurposeLiver metastases confer poor outcomes and attenuate the benefit of immunotherapy across solid tumors. This study investigated how the hepatic metastatic niche promotes CD8 T cell dysfunction and immunotherapy resistance in small-cell lung cancer (SCLC). Experimental DesignClinical outcomes and tumor gene expression were integrated with multi-region single-cell RNA sequencing of T cells from rapid-autopsy SCLC metastases, together with spatial transcriptomics. SCLC-hepatocyte conditioned-media models were combined with stable-isotope tracing, mass spectrometry, functional and metabolic assays, and ChIP-qPCR to define mechanisms of CD8 T cell suppression. ResultsLiver metastases were associated with inferior survival and reduced benefit from immune checkpoint blockade. Multi-region single-cell analysis showed that CD8 T cells from liver metastases exhibited an exhaustion-associated state enriched for hypoxia, lactate, and TGF-{beta} programs. SCLC-hepatocyte crosstalk generated a lactate- and TGF-{beta}-rich microenvironment that reduced CD8 T cell effector function, proximal T cell receptor signaling, glycolytic fitness, viability, and proliferation. Stable-isotope tracing demonstrated transfer and accumulation of co-culture-derived lactate in recipient CD8 T cells, with limited entry into downstream pyruvate-linked pathways. Lactate accumulation was accompanied by increased H3K18 lactylation at the PDCD1, LAG3, and TGFB1 regulatory loci. In parallel, SCLC-hepatocyte crosstalk increased paracrine TGF-{beta} and activated canonical SMAD2 signaling in CD8 T cells. TGF-{beta} receptor inhibition restored CD8 T cell proliferation. In the phase III IMpower133 cohort, a combined lactate-TGF-{beta} transcriptional program was associated with inferior survival, most strongly in patients with liver metastases. ConclusionsTumor-hepatocyte crosstalk generates convergent lactate and TGF-{beta} signals that drive CD8 T cell dysfunction in liver metastases. This hepatic immune-metabolic circuit provides a potential mechanism for immunotherapy resistance and supports therapeutic strategies targeting TGF-{beta} signaling in liver-metastatic SCLC. Translational RelevancePatients with SCLC liver metastases have poor outcomes and derive limited benefit from immune checkpoint blockade, but actionable mechanisms of hepatic immune resistance remain undefined. We identify an immune-metabolic circuit in which SCLC-hepatocyte crosstalk generate lactate and TGF-{beta} signals that converge on CD8 T cells. Stable-isotope tracing demonstrates the transfer and accumulation of tumor-hepatocyte-derived lactate in recipient T cells, which causes H3K18 lactylation at exhaustion- and TGFB1-associated loci. In parallel, paracrine TGF-{beta} activates canonical SMAD signaling and reinforces proliferative dysfunction. TGF-{beta} receptor inhibition restores CD8 T cell proliferation. In the phase III IMpower133 cohort, a combined lactate-TGF-{beta} program is associated with inferior survival, particularly among patients with liver metastases. These findings provide a mechanistic and biomarker framework for testing TGF-{beta}-directed strategies in liver-metastatic SCLC, a population with substantial unmet clinical need.

cancer biology↗

bioRxiv2026

Hepatobiliary Progenitor-like Reprogramming in Liver Metastases

Metastatic progression requires cancer cells to adapt to the unique constraints of distant organ microenvironments, yet the mechanisms that drive organ-specific adaptations remain poorly understood. Here, we show that the liver actively rewrites metastatic cancer cell identity, driving tumor cells toward a hepatobiliary progenitor-like state. Through integrated transcriptomic, proteomic, metabolomic, and epigenomic analyses of patient-derived rapid-autopsy samples and experimental models, we identify this state as selectively enriched in liver metastases. It is characterized by co-activation of hepatic and biliary/progenitor regulators HNF4A and SOX9 and is observed across multiple epithelial cancers, indicating a conserved response to the hepatic niche. Mechanistically, hepatocyte-derived TGF-{beta} and hypoxia converge to activate a HIF-1-ACLY axis, increasing nuclear acetyl-coenzyme A availability and histone acetylation at hepatic lineage regulatory elements to drive hepatobiliary reprogramming. This coordinated niche-response program can be captured transcriptionally and is associated with inferior overall survival. Disruption of this pathway suppresses hepatic reprogramming and impairs liver metastatic fitness. These findings identify the liver as an active determinant of metastatic cell fate, linking microenvironmental signaling to metabolic and chromatin remodeling programs that enable lineage plasticity. More broadly, they reveal organ-specific reprogramming as a fundamental principle of metastasis and a therapeutic vulnerability in liver metastases.

cancer biology↗

bioRxiv2026

Microelectrode arrays enable directional stereo-EEG during kainate-mediated seizures

Surgical planning for drug-resistant epilepsy often relies on stereo-EEG (sEEG) recordings obtained with cylindrical ring electrodes. Prior modeling studies, including lead-field analysis, suggest that microelectrodes distributed around an sEEG-sized insulating body offer superior source amplification and directional sensitivity that are not available with either a ring design or microelectrodes on micro-structures, e.g. Neuropixel. However, these advantages have not been demonstrated in seizure models. This study evaluated high-density sEEG recordings using directional microelectrode arrays in a kainate-mediated rat model (n=6). Two 64-channel microelectrode arrays were implanted near the hippocampus, and the signals were spatially averaged to emulate virtual ring electrodes for comparison. Device locations were reconstructed and placed in copies of the Waxholm Space Rat Brain Atlas registered to subject-specific MRI scans. In subjects exhibiting seizures (n=4), automated line length detection showed that microelectrode signals identified epileptiform activity sooner and with greater specificity than ring electrodes. In subjects that only seized post-kainate injection (n=3), manual review by a board-certified epileptologist confirmed that microelectrodes provided the earliest onset detection times. Furthermore, the microelectrode arrays high resolution revealed distinct instances of hyperactivity occurring at similar depths but from different directions -- a feature indistinguishable to standard ring electrodes. These results demonstrate that microelectrodes on a large insulating body significantly enhance signal quality and spatial localization. This technology offers a potential advancement over current clinical standards for identifying seizure foci during surgical planning. SIGNIFICANCESurgical treatment for drug-resistant epilepsy depends on accurately identifying the brain regions where seizures begin. Current stereo-EEG electrodes sample activity with ring contacts that average signals around the probe shaft, potentially obscuring directional differences in nearby neural activity. This study shows that microelectrodes distributed around an sEEG-sized insulating body can improve seizure-related signal detection and spatial localization compared with ring-like recordings from the same implant locations. By resolving activity that conventional ring electrodes cannot distinguish, high-density directional sEEG may provide more informative recordings for seizure mapping. These findings support the development of next-generation intracranial electrodes for improving epilepsy surgical planning.

neuroscience↗

bioRxiv2026

An autoinhibitory regulatory switch governs heterotypic phase separation and liquid-to-solid phase transition of TDP-43 and tau into cytotoxic amyloids

Biomolecular condensates formed via macromolecular phase separation of proteins and nucleic acids control a myriad of essential cellular processes, whereas abnormal phase transitions into solid-like aggregates are associated with a range of fatal neurodegenerative diseases. Here, we present a unique case to demonstrate that two neuronal proteins, TDP-43 and tau, undergo heterotypic phase separation via domain-specific interactions regulated by an autoinhibitory conformational switch. Using single-molecule FRET (Forster resonance energy transfer), in combination with multi-color high-resolution imaging, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, single-droplet fluorescence anisotropy imaging, homoFRET microscopy, fluorescence lifetime-FRET imaging, vibrational Raman spectroscopy, and electron microscopy, we unmask the interplay of molecular drivers and dissect the sequence of events associated with the formation of TDP-43:tau co-condensates that undergo liquid-to-solid phase transitions into cytotoxic amyloids. Our cellular studies show that a disease-associated cytosolic fragment of TDP-43 recruits tau into oxidative stress-induced cytoplasmic granules, eliciting cellular toxicity. Our findings provide mechanistic underpinnings of co-condensation-mediated aberrant phase transitions associated with exacerbated neuropathological outcomes.

biophysics↗

bioRxiv2026

ACTIVATED CASO₄-INDUCED VACUOLATION AS A QUANTITATIVE PLATFORM FOR PHAGOCYTOSIS-DRIVEN DRUG SCREENING

Cytoplasmic vacuolization is a fundamental process associated with phagocytosis, lysosomal acidification, and autophagy, yet robust in-vitro models for its quantification and pharmacological screening remainlimitedor insufficiently established. In this study, we demonstrate that thermally activated calcium sulfate (ACS) induces extensive vacuolation across mammalian cell lines including HeLa, RAW 264.7, 3T3-L1, and SH-SY5Y, thereby establishing a versatile platform to study vacuole biogenesis. To ensure reproducibility, particle heterogeneity was addressed using sedimentation-based fractionation, with homogeneous suspensions obtained at the 5th minute producing stable and consistent vacuole formation. Vacuolation was subsequently quantified by Neutral Red (NR) uptake, dose and time dependent response analyses confirmed direct correlation between ACS concentration and vacuole induction. The assay was validated with bafilomycin A1 (BFA1), a selective V-ATPase inhibitor, which served as a positive control and demonstrated concentration and time dependent inhibition of vacuole formation and acidification. Building on this framework, ten commercially available drugs were screened, revealing distinct profiles ranging from early cytotoxicity, strong vacuole inhibition to partial suppression or negligible effects. This dual capacity to discriminate between vacuole inhibition and cytotoxic responses highlights the utility of ACS-induced vacuolization as a sensitive and scalable in vitro platform. Collectively, our findings position this system as a tractable assay for mechanistic studies of vacuole biology and a functional screening tool for identifying modulators of lysosomal and phagocytic pathways relevant to infection, Lysosomal Disorders, and Phagocytotic dysfunction disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/711143v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@e5b920org.highwire.dtl.DTLVardef@1dd1072org.highwire.dtl.DTLVardef@62ae59org.highwire.dtl.DTLVardef@a468bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2026

Diversified Portfolios of Bacterial Communities in Peri-urban Miyawaki Forests and Grassland Shaping Ecosystem Functions and Services

Soil microflora is fundamental to ecosystem functioning, yet their contribution in Miyawaki afforestation, a globally implemented ecological engineering approach, remains poorly characterized. In the present study, we examined the bacterial taxonomic diversity and their functional potential in a peri-urban Miyawaki mini-forest and compared with a nearby grassland the pre-existing ecosystem across dry and wet seasons. The Miyawaki plantation comprised of highly diverse native trees, sub-trees and shrubs spanning evergreen and deciduous varieties, potentiating nitrogen-fixation, diverse litter generation and rooting strategies resulting in pronounced functional heterogeneity. Notably relative to grassland, the Miyawaki forest was intensively managed and supplemented with organic amendments, and supportive irrigation, buffering the seasonal moisture stress. Using 16S rRNA amplicon sequencing of soil eDNA, we characterized seasonal variation in soil bacterial communities in both the systems. The observed soil bacterial community organization in forest as compared to the unmanaged grassland indicates combined influence of vegetation structure, dense canopy cover, continuous litter generation and root exudates. Microbial assemblages in the forest specialised in heterotrophic complex carbon degradation, biofilm formation, exopolysaccharide production and sporulation pathways which suggests adaptive abilities to anoxic microsites and other stressful conditions. In contrast, grassland soils harboured less diversified bacterial communities dominated by phototrophic and oxidative stress adaptation pathways consistent with sun lit, non-irrigated and moisture-variable conditions. Nonetheless, functional divergence in dry season reflects temporal reorganization of microbial communities marking a gradual trend towards soil ecosystem development. Together, these findings establish microbial baseline for Miyawaki forests revealing how tree-dense mini-forests restructure soil bacterial communities relative to grasslands highlighting the value of identifying soil microbial indicators for critically evaluating urban afforestation outcomes over extended time scales to inform sustainable design and policy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/704982v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@15e1bb1org.highwire.dtl.DTLVardef@16c1dddorg.highwire.dtl.DTLVardef@11c99ecorg.highwire.dtl.DTLVardef@bd6d1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic representation of the study depicting the Miyawaki forest and nearby grassland.

ecology↗

bioRxiv2026

In virio secondary RNA structure analysis of influenza A virus

The genome of influenza A viruses (IAV) consists of eight distinct gene segments that need to be packaged into new virions. Although this process is thought to be controlled by specific RNA-RNA interactions between segments, few studies have identified the RNA structure inside virions and performed functional validations on these structures. Using high-throughput probing techniques such as SHAPE-MaP and DMS-MaPseq, we mapped the secondary structure of the A/Puerto Rico/8/1934 (PR8) IAV genome. We discovered 173 putative structural motifs in both packaging and central regions of all segments. Fifteen motifs were selected for functional studies by introducing synonymous mutations to destabilize the predicted structure and assessing viral fitness. Synonymous mutations in three predicted RNA structures within the packaging signals of NP and PB1 attenuated virus replication in vitro. Interestingly, combining structural changes in multiple motifs within the packaging signals enhanced viral attenuation, supporting the idea of synergistic structural contributions to viral fitness. In contrast, structural synonymous mutations in RNA secondary structures outside packaging signals had no impact on virus replication in vitro. Overall, our findings revealed several new RNA secondary structures within the packaging signals of IAV gene segments that play a critical role in the IAV life cycle.

microbiology↗

bioRxiv2026

ImmunoPheno: A Computational Framework for Data-Driven Design and Analysis of Immunophenotyping Experiments

Immunophenotyping is fundamental to characterizing tissue cellular composition, pathogenic processes, and immune infiltration, yet its accuracy and reproducibility remain constrained by heuristic antibody panel design and manual gating. Here, we present ImmunoPheno, an open-source computational platform that repurposes large-scale single-cell proteo-transcriptomic data to guide immunophenotyping experimental design and analysis. ImmunoPheno integrates existing datasets to automate the design of optimal antibody panels, gating strategies, and cell identity annotation. We used ImmunoPheno to construct a harmonized reference (HICAR) comprising 390 monoclonal antibodies and 93 human immune cell populations. Leveraging this resource, we algorithmically designed minimal panels to isolate rare populations, such as MAIT cells and pDCs, which we validated experimentally. We further demonstrate accurate cell identity annotation across publicly available and newly generated cytometry datasets spanning diverse technologies, including spatial platforms like CODEX. ImmunoPheno complements expert curation and supports continual expansion, providing a scalable framework to enhance the accuracy, reproducibility, and resolution of immunophenotyping.

bioinformatics↗

bioRxiv2026

An anatomical hotspot for striatal dopamine-acetylcholine interactions during reward and movement

Dopamine (DA) and acetylcholine (ACh) are key neuromodulators that regulate striatal circuits underlying movement and reinforcement learning. Evidence suggests that DA and ACh systems interact, but where and how interactions are expressed across striatal regions in behaviorally relevant release dynamics remains unknown. We applied micro-fiber arrays to simultaneously measure striatum-wide DA and ACh in behaving mice, revealing an anatomical organization in which DA-ACh anti-correlations were concentrated in a hotspot in the anterior dorsolateral striatum (aDLS). Anti-correlations resulted from temporally coincident pairs of spontaneous and event-locked transient peak and dip events occurring in a DA[->]ACh sequence. The aDLS localized hotspot was consistently expressed within distinct signals associated with unpredicted rewards, learned and extinguished Pavlovian cues, and locomotion initiation and invigoration phases, for which we revealed novel, opposing DA-ACh dynamics. Optogenetic activation of DA neurons selectively suppressed spontaneous ACh release within the aDLS hotspot, and ex vivo recordings revealed enhanced D2-mediated ACh inhibition in aDLS relative to ventral regions, suggesting a mechanistic basis for this spatial specificity. These findings demonstrate that DA-ACh interactions during behavior are spatially organized, rather than uniformly conserved, and shape behaviorally relevant dynamics to regulate region-specific functions in learning and movement control.

neuroscience↗

bioRxiv2026

Meta-learning is expressed through altered prefrontal cortical dynamics

Learning where and when rewards like food and water are available is essential for survival1,2. In the simplest cases where resource availability is stable, animals can learn reward contingencies by integrating outcomes across repeated samples of each possible action. In more natural settings, however, reward availability is governed by structured higher-order rules such as depletion and repletion over time. To adapt flexibly to such changing environments, optimal choices require meta-learning wherein animals learn how to learn from external feedback, ultimately enabling them to infer the underlying reward structure from abstract, generalizable rules rather than relying solely on recent outcomes3,4. The existence of meta-learning in animal behavior is well established3-8, yet the neural circuits and computations that implement it remain poorly understood9-11. Here we investigated meta-learning using a spatial foraging task in which rats acquired a depletion-repletion rule that regulated reward availability, and carried out longitudinal, high-density recordings from the medial prefrontal cortex (mPFC). We show that meta-learning engages specific, systematic changes in mPFC neural dynamics that embed the learned rule and thereby alter how the network learns action values from reward outcomes. These dynamics are based on mixed coding of task structure and value in individual mPFC neurons. At the population level, this coding organizes into low-dimensional dynamical motifs that generalize across task conditions. As meta-learning progresses, these motifs are reshaped to instantiate both rule-guided inference of future states before outcome delivery and rule-based value updating during the outcome period. These results indicate that meta-learning sculpts pre-existing prefrontal dynamics to support the acquisition of new, generalizable reward-learning strategies.

neuroscience↗

bioRxiv2025

Meditation as a Bioactive Intervention: Molecular and Neurophysiological Mechanisms Revealed by Connectivity Mapping

Meditation practices are often used as non-pharmacological adjunct therapy for managing stress and well-being benefits. We propose that beneath their non-pharmacological facade, meditation practices might operate via drug target modulation having profound neurophysiological effects. Firstly, we leverage the Connectivity Map (CMap) to investigate (a) the overlap between meditation-induced molecular signatures and established drug responses, and (b) the pathways and mechanisms contributing to meditation potential therapeutic effects. This was studied in a comprehensive temporal RNAseq dataset comprising premeditation, meditation, and follow-up stages from a clinical trial involving 106 participants practising inner engineering meditation. Meditation signatures overlapped with over 438 drugs, but predominantly with drugs targeting the neuroactive ligand receptor pathways, capable of modulating the brains excitation-inhibition (E/I) balance. Next, to bridge the meditation-induced molecular changes to measurable neurophysiological effects, we performed a comprehensive meta-analysis of drugs affecting E/I balance through the lens of non-invasive brain stimulation, i.e., transcranial magnetic stimulation (TMS-EMG and TMS-EEG). Using multiple correspondence analysis, we then clustered the CMap informed neuroactive drugs and their E/I effects on the same latent space. We found that meditations effects mimic drugs targeting the GABAergic, Glutametergic and other neuromodulatory system pathways. Our findings lead to a working model to objectively test neurophysiological changes resulting from meditation that can lead to evidence-based clinical applications. Overall, these findings support the view that meditation acts as a biologically active intervention capable of modulating molecular pathways and cortical excitability, rather than functioning solely as a psychological or contemplative practice.

systems biology↗