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Genetic Disruption at the CIP2A Locus Modulates T Cell Responses and Attenuates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) driven by pathogenic T cell-mediated inflammation. Fingolimod (FTY720), an approved therapy for MS, is an established activator of protein phosphatase 2A (PP2A). However the contribution of PP2A in autoimmune neuroinflammation remains incompletely understood. Here, we addressed this question using experimental autoimmune encephalomyelitis (EAE), a murine model of MS, in mice carrying a genetic disruption of the locus encoding cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A. Mice with disruption of the CIP2A locus, the knock out (KO) mice, exhibited attenuated EAE severity compared with wild-type (WT) controls. Histological and flow-cytometric analyses revealed markedly reduced infiltration of mononuclear cells, including CD4 and CD4CXCR6 encephalitogenic T cells, in the CNS of diseased KO mice. Reduced numbers of these T cell populations were also observed in peripheral lymphoid organs of the Cip2a-deficient mice during EAE, while T cell abundance was comparable under steady-state conditions, suggesting impaired activation-induced expansion rather than altered homeostasis or migration. Single-cell RNA sequencing of CNS and lymph node immune cells revealed changes in cell-type abundance and gene expression. Notably, Il17a expression was reduced in CNS CD8+ T cells and showed a similar trend in {gamma}{delta} T cells. Together, our findings reveal that genetic disruption at the CIP2A locus attenuates EAE, possibly by limiting the expansion and accumulation of encephalitogenic T cell populations in CNS. These results identify the CIP2A locus as a previously unrecognized regulator of T cell-driven autoimmune neuroinflammation and provide new insights into mechanisms that restrain pathogenic T cell responses during EAE.

immunology

NAE1-Dependent Protein Neddylation Preserves Endothelial Identity and Vascular Integrity

Background: Endothelial dysfunction is a central driver of cardiovascular and inflammatory diseases, yet the post-translational mechanisms that preserve endothelial homeostasis remain incompletely understood. Protein neddylation, the covalent conjugation of a ubiquitin-like modifier, regulates diverse cellular processes, yet its physiological role in the vascular endothelium remains unknown. This study investigated whether protein neddylation is required to preserve endothelial identity and vascular homeostasis. Methods: We generated tamoxifen-inducible endothelial-specific Nae1 knockout mice to inhibit neddylation and combined bulk RNA sequencing, single-cell and single-nucleus transcriptomics, quantitative proteomics, biochemical analyses, and gain- and loss-of-function approaches to define the role of endothelial neddylation in vascular homeostasis and inflammatory injury. Results: Endothelial-specific Nae1 deletion caused rapid mortality associated with vascular leakage, platelet accumulation, inflammation, and multi-organ injury. Multi-omics analyses demonstrated profound loss of endothelial identity, characterized by suppression of core endothelial programs and activation of inflammatory, procoagulant, and pyroptotic pathways. Single-cell analyses revealed progressive endothelial dysfunction culminating in depletion of the endothelial population and remodeling of the vascular niche. Mechanistically, endothelial neddylation deficiency activated gasdermin D (GSDMD)- and gasdermin E (GSDME)-dependent pyroptosis, whereas dual inhibition of GSDMD and GSDME markedly attenuated inflammatory transcriptomic remodeling, vascular injury, hepatocyte death, immune cell infiltration, and platelet accumulation. Translational analyses demonstrated reduced endothelial neddylation in experimental endotoxemia and decreased expression of neddylation pathway components in human atherosclerosis and COVID-19 datasets. Conversely, restoration of endothelial neddylation partially reversed inflammatory endothelial transcriptomic reprogramming in vivo. Conclusions: NAE1-dependent protein neddylation is an essential regulator of endothelial identity and vascular integrity. Loss of endothelial neddylation promotes gasdermin-dependent pyroptosis and thrombo-inflammatory vascular injury, whereas restoration of the neddylation pathway mitigates inflammatory endothelial dysfunction. These findings identify endothelial neddylation as a fundamental mechanism maintaining vascular homeostasis and a potential therapeutic target for cardiovascular and inflammatory diseases.

pathology

Replication stress at centromeres biases the segregation of DNA damage

Replication-associated errors can cause DNA damage to accumulate on the newly synthesized strand over time. In specific cases such as stem cells, retention of the immortal strand used as template preserves one daughter cell into pluripotency while correlating with terminal differentiation of the damage one. In somatic cells, DNA damage distribution after mitosis remains unclear. Here, we uncovered a mechanism of non-random segregation of the DNA damage marker gH2AX occurring during a single cell division cycle. Replication stress using hydroxyurea (HU) upon release into S phase in RPE-1, BJ, hCEC D29 and fibroblasts showed reproducible Non-Random Segregation (NRS) of gH2AX in the ensuing G1, a phenotype not observed in any of the cancer cell lines analyzed. Notably, removal of R-loops led to a reduction of cells with NRS, whether RNaseH1 was over-expressed globally or exclusively targeted to centromeres, indicating that centromeric DNA-RNA hybrids contribute to NRS of the damage. In line with our previous evidence of centromeric chromatin disruption leading to R-loops, rapid removal of the histone H3 variant CENP-A causes damage and NRS, although to a lower extent than HU alone. This implies that additional mechanisms contribute to centromeric R-loops and NRS of damage in the daughter cells upon mitotic exit. Mechanistically, chemical inhibition of the catalytic activity of Rad51 led to a significant drop in NRS without a change in the total amount of damaged cells, implying involvement of the Homologous Recombination (HR) pathway to accumulation of gH2AX to only one chromatid. In turn, this affects the spindle-kinetochore with a measurable length asymmetry, inducing mechanical and/or epigenetic signals that affect the orientation of the sister chromatids on the metaphase plate to bias segregation. Altogether, we found replication-induced asymmetric segregation of DNA damage during mitosis that is influenced by centromeric R-loops, Rad51 activity and spindle dynamics, with implications on cell fate, chromosome and genome stability in the daughter cells.

cell biology

Readout-dependent time-of-flight weighting of absorption and flow in interferometric diffuse optics

Joint optical measurement of hemoglobin and blood-flow dynamics could provide a compact route to richer functional monitoring of tissue physiology. However, hybrid diffuse-optical systems commonly optimize photon time-of-flight (TOF) for individual readouts, even when absorption and flow are recovered from the same acquisition. Here, we asked whether a single TOF window can optimally preserve both signals. Dual-wavelength TOF-iSCOS recovered absorption, hemoglobin and relative blood-flow index (BFI) from the same interferometrically sensed optical field, while Early, Middle and Late photon windows provided progressively greater weighting of longer and deeper photon paths. During forearm cuff occlusion in 11 adults, absorption and flow showed markedly different TOF dependence: Late-gate BFI responses were retained, whereas absorption decreased to a median Late/Early ratio of 0.18. The resulting readout-by-TOF interaction was positive in all 11 participants (median 2.61 octaves, 95% bootstrap CI 2.44-3.22; exact sign-test P = 9.8 x 10-4). Readout-specific gate selection increased held-out response retention by 24.8% (95% CI 8.8-42.5%) relative to a single common gate. We then tested the approach during prefrontal working-memory activation in seven participants using a higher-load 2-back task relative to a low-load 0-back control. Late-gate BFI increased in all seven participants (+5.07%, 95% CI 1.46-8.68%; P = 0.014) and remained positive after adjustment using an Early-TOF signal, whereas hemoglobin responses were less consistent. Layered Monte Carlo modeling showed that the same detected photon histories acquire different absorption and dynamic-scattering sensitivity across TOF, linking the experimental effects to anatomy- and geometry-conditioned tissue weighting. Within the present geometry and photon budget, the central result is therefore not that one readout is intrinsically superior, but that absorption and flow recovered from the same optical field retain physiological information differently across photon time-of-flight.

bioengineering

Systems-level proteomic reprogramming reveals mitochondrial restoration and inhibition of Rho GTPase-mediated cytoskeletal and inflammatory signaling in CKD

Chronic kidney disease (CKD) is a progressive disorder characterized by metabolic dysfunction, mitochondrial impairment, oxidative stress, and chronic inflammation, ultimately leading to irreversible renal damage. Despite advances in understanding CKD pathophysiology, effective therapies targeting these interconnected molecular processes remain limited. In this study, we performed a comprehensive data-independent acquisition (DIA)-based proteomic analysis to investigate the molecular alterations associated with CKD and to evaluate the therapeutic impact of DVA treatment. Using a CKD model with three treatment conditions (DVA, KY, and DVA+KY) alongside disease and healthy controls, we quantified global proteomic changes and applied statistical filtering (fold change [≥]2, p [≤]0.05) followed by K-means clustering (k=10). Distinct protein clusters revealed bidirectional modulation upon DVA treatment. Notably, Cluster 1 comprised proteins downregulated in CKD but significantly restored following DVA administration, while Cluster 2 included proteins elevated in CKD that were suppressed by DVA. Pathway enrichment and network analyses demonstrated that Cluster 1 proteins were predominantly associated with mitochondrial function, oxidative phosphorylation, and metabolic processes, whereas Cluster 2 proteins were enriched in immune signaling, oxidative stress, cytoskeletal remodeling, and proteostasis pathways. At the molecular level, DVA treatment restored key mitochondrial and metabolic regulators, including components of the electron transport chain (e.g., COX5A, NDUFS5, SDHB) and redox homeostasis proteins, indicating recovery of cellular bioenergetics. Concurrently, DVA suppressed inflammatory mediators (STAT2, IFI47, GBP2), oxidative stress-related proteins (CYBB, PRDX5), and cytoskeletal regulators linked to renal injury (ARHGEF12, FMNL2). Network and Reactome analyses further confirmed coordinated modulation of interconnected biological systems rather than isolated protein changes. Collectively, our findings demonstrate that DVA exerts a dual therapeutic effect by restoring essential mitochondrial and metabolic pathways while simultaneously suppressing inflammation, oxidative stress, and cytoskeletal dysregulation in CKD. This systems-level proteomic reprogramming highlights DVA as a promising candidate for CKD intervention and provides mechanistic insights into disease progression and therapeutic targeting.

systems biology

Mapping Light-Induced Conformational Dynamics of Pigeon Cryptochrome 4 by HDX-MS: Structural Transitions from Spin Pair Formation to Activated Conformational States

The navigational prowess of migratory birds is thought to arise from light-dependent radical-pair chemistry in cryptochrome 4 (CRY4), yet the slow structural transitions that couple photochemistry to signaling remain elusive. Here, we combine temperature-controlled steady-state UV-visible spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate the photochemical and conformational dynamics of pigeon CRY4 (ClCRY4). Steady-state measurements at 5-25 {degrees}C reveal that lower temperatures slow FAD photoreduction and prolong the FAD neutral semiquinone signaling state. This occurs without a solvent kinetic isotope effect, implicating a conformational change rather than proton transfer as the rate determining step in FAD neutral semiquinone formation. Simultaneous HDX-MS under blue-light exposure identifies protection near the FAD-binding site and C-terminal region. To enhance sensitivity, we developed a pump-probe HDX-MS approach at 10 {degrees}C. This reveals eight peptides (within the phosphate-binding loop, protrusion motif, electron-transfer-chain loops and C-terminal tail) that exhibit rapid ([≤]10 s) and sustained light-induced protection, delineating early conformational rearrangements as a prerequisite for FAD neutral semiquinone accumulation. The findings of slower onset HDX protection as well as a bimodal pattern of deuterium uptake in the phosphate-binding loop further identify a local redistribution of conformational substates on the time scale of the accumulation of the signaling species. Site specific mutagenesis within the CTT supports the findings, which lead to a model in which blue light triggers rapid clamping down of protein near the two regions of spin pair separation, followed by a rate limiting closure of a surface loop. The resolution of time-dependent structural transitions that follow photoactivation of CRY4 resolves the interface between quantum radical-pair formation and classical conformational changes, while providing an enhanced structural framework for the molecular events that underlie avian magnetoreception.

biophysics

Development and pharmacological evaluation of an intranasal liposomal norbinaltorphimine formulation for the prevention of pain-induced negative affect

Chronic pain is frequently accompanied by negative affect and motivational deficits due to dysregulated mesocorticolimbic dopamine and kappa opioid receptor (KOR) signalling. Although intracranial KOR antagonism prevents pain-induced negative affect in preclinical models, systemic KOR antagonists can produce adverse off-target effects in the periphery, thereby limiting its clinical utility. Consistent with this, we found that systemic administration of KOR antagonist norbinaltorphimine (NorBNI), exacerbated motivational deficits in rats with persistent inflammatory pain. We hypothesized that maximizing central and minimizing peripheral KOR antagonism could overcome these limitations. To test this, we engineered an intranasal liposomal NorBNI formulation incorporated into an in-situ forming mucoadhesive hydrogel to enable selective nose-to-brain delivery (Nor-BNILV-HG). We characterized its physicochemical properties and functional efficacy in rats with inflammatory pain produced by Complete Freund's Adjuvant (CFA). NorBNI-loaded liposomes exhibited high drug entrapment efficiency, nanometric size, and suitable surface charge for intranasal administration. The selected thermosensitive hydrogel demonstrated appropriate gelation properties and sustained drug release. Intranasal administration of NorBNI-LV-HG produced negligible systemic NorBNI levels compared with intraperitoneal delivery. In vivo microdialysis showed that NorBNI-LV-HG prevented KOR agonist-induced reductions in nucleus accumbens (NAc) dopamine release, confirming functional central KOR blockade. Behaviourally, intranasal NorBNI-LV-HG attenuated pain-induced impairments in sucrose motivation. Importantly, unlike systemic NorBNI, repeated intranasal NorBNI-LV-HG did not alter mechanical nociceptive thresholds in pain-naive animals, suggesting this strategy mitigates unwanted peripheral nociceptive effects. Together, these findings demonstrate that intranasal NorBNI-LV-HG achieves functional brain KOR antagonism while minimizing systemic exposure and off-target effects. Selective nose-to-brain delivery of KOR antagonists therefore represents a promising therapeutic strategy to prevent and potentially reverse the affective and motivational consequences of pain and may overcome key translational barriers associated with systemic KOR treatments.

neuroscience

A mechanistic basis for CD8+ T cell expansion sensitivity as a predictor of HIV post-treatment control

A key goal in HIV-1 cure research is to understand why some individuals control viral rebound after stopping antiretroviral therapy (ART). Recent human studies have identified responding CD8+ T cells expressing Ki-67 and the transcription factor TCF-1 as correlates of post-treatment control, but the mechanistic basis of this association remains unclear. Using the theoretical framework of Conway and Perelson, we fit mechanistic within-host models to viral load and CD8+ T cell data from 9 individuals in a combination immunotherapy trial following ART interruption. Although Ki-67 and TCF-1 measurements were not used for fitting, the inferred effector cell expansion sensitivity, i.e., the responsiveness of effector expansion to low antigen levels, shows a strong linear relationship with Ki-67 and TCF-1 levels at rebound (Pearsons r {approx} 0.8). Building on this, we show analytically that the post-rebound viral load set point is inversely proportional to the effector cell expansion sensitivity, and thus strongly correlates with cycling (Ki-67+) CD8+ T cells (r {approx} -0.8) at rebound, and a subset that expresses TCF-1 (r {approx} -0.9). In effect, individuals with a larger proportion of CD8+ T cells responding to viral rebound, and a greater representation of TCF-1 expressing cells within the responding subset, achieve markedly lower viral set points through a higher effector cell expansion sensitivity. This mechanism is consistent with prior modeling in a non-intervention ATI setting, suggesting it may generalize across more rebound contexts. Our results provide a mechanistic explanation why both Ki-67+ responding CD8+ T cells and their TCF-1-expressing subset predict post-treatment control, linking clinical correlation to its underlying cause and highlighting Ki-67 and TCF-1 as potential early biomarkers of HIV immunotherapy success.

immunology

A mouse-adapted Staphylococcus aureus strain enables lifelong neonatal colonization and elicits a Th17-dominated immune response

The opportunistic pathogen Staphylococcus aureus persistently colonizes the anterior nares of up to 20% of the human population, yet there were no persistent mouse colonization models to study host-pathogen interaction. Using the mouse-adapted S. aureus strain JSNZ (CC88-MSSA), we established a neonatal S. aureus colonization model in C57BL/6N mice. Natural neonatal colonization was achieved by vertical transmission in a JSNZ-positive breeding colony. Offspring were followed for up to 69 weeks and found persistently colonized in the nose and cecum with high bacterial loads. Adult mice were colonized by intranasal inoculation of JSNZ; controls received PBS. The colonization patterns and the S. aureus-specific T cell responses were then monitored over a period of 28 days and compared between age-matched mice colonized as neonates or adults. The neonatal group remained persistently colonized in nose and gut with high bacterial densities. In contrast, mice colonized as adults had lower and declining bacterial loads in the nose. Some eliminated S. aureus from the nares, while all remained colonized in the gut. Neonatally colonized mice exhibited reduced nasal chemokine levels, which may have favored the prolonged S. aureus persistence. Ex vivo re-stimulation of cervical lymph node cells with an S. aureus antigen cocktail revealed a Th17-dominated antigen-specific T cell response in both colonized groups. The lymph node cells secreted large amounts of IL-17, but Th1-, Th2-associated and regulatory cytokines were also detected. The cytokine patterns were similar in both colonized groups except for IL-5, which was more abundant upon neonatal colonization. In conclusion, vertical transmission of the mouse-adapted S. aureus strain JSNZ reliably establishes persistent high-density neonatal colonization, providing a physiologically relevant model for the study of S. aureus host interactions. Route and timing of colonization do not fundamentally affect the T cell response to S. aureus.

immunology

Vitamin D counters bone invasion by mammary cancer through inhibition of inflammation and epithelial-to-mesenchymal transition

Vitamin D deficiency is associated with poor outcome in several cancers in humans, and administration of vitamin D or analogs has been shown to decrease tumor progression and metastasis in animal mammary cancer models. We previously demonstrated significant acceleration of carcinogenesis in vitamin D-deficient mouse mammary tumor virus-polyoma middle T (MMTV-PyMT) mammary cancer model as well as of its spontaneous metastasis to lungs. While vitamin D also plays a role in skeletal metastasis, detailed mechanisms of its promotion of bone invasion and metastatic events are not completely elucidated. In the present study we used tibially-injected MMTV-PyMT mammary tumor cells to analyse how dietary-induced vitamin D deficiency in non-immunodeficient FVB mice accelerates bone invasion. Mechanistically, we observed vitamin D deficiency to increase pro-inflammation cytokines and nestin expression in internal bone surface and marrow, and to increase epithelial-to-mesenchymal transition (EMT) through Zeb1 transcription factor. In vitro, treatment of MMTV-PyMT tumor cells with CXCL12 was observed to stimulate Zeb1 expression, and this effect was efficiently countered by 1,25(OH)2D treatment. Analysis of cytokines in MMTV-PyMT mammary tumor cells in vitro showed significant reduction in several pro-inflammatory cytokines with 1,25(OH)2D treatment (GM-CSF, ICAM-1, IL-1ra, IP-10, JE, MCP-5, MIP-1, MIP-1{beta}, MIP-2, RANTES and CXCL12), a crucial observation in view of the current evidence that inflammation is one of the hallmarks of cancer. Furthermore, vitamin D repleteness is associated with very high expression of Socs1 (suppressor of cytokine signalling 1), an inhibitor of JAK/STAT pathway which prevents excessive inflammatory responses and has a tumor-suppressive role. These findings provide a strong link between vitamin D deficiency and acceleration of inflammation-driven bone invasion, and nestin and EMT. The evidence suggests that vitamin D-repleteness in breast cancer patients could enhance the efficacy of co-administered therapies in preventing invasion of skeletal sites.

cancer biology

Stepping-stone population structure and sidespread clonality of the mesophotic octocoral Swiftia exserta in the warm temperate northwest Atlantic

Isolated mesophotic banks form spatially discrete networks of patchy ecosystems along continental shelves. Their long-term persistence depends on connectivity among populations of the dominant coral species that structure these communities. Yet the scale of larval-mediated gene flow across these networks remains poorly characterized. Similarly, the extent to which clonal propagation shapes local population dynamics is unresolved, leaving the relative contributions of sexual and asexual reproduction to population maintenance an open question. Here, population genomics and high-resolution (1km) biophysical larval dispersal modeling are integrated to resolve the genetic connectivity and clonal dynamics of Swiftia exserta, a key habitat-forming octocoral, across 13 mesophotic banks spanning the U.S. Gulf and eastern coast within the Warm Temperate Northwest Atlantic (WTNWA) coastal and shelf biogeographic province. Genome-wide SNP markers derived from Restriction-site Associated Sequencing (RADseq) were genotyped across 288 individuals. About 40% of individuals belonged to clonal genets confined to single banks within meters of one another, indicating that asexual propagation sustains local density but plays no detectable role in inter-bank connectivity. Population genetic analyses pointed to isolation by distance as the primary structuring force, with the majority of molecular variance residing within rather than among populations. The west-most population, East Flower Garden Banks, was the most differentiated population in the WTNWA Northern Gulf ecoregion, reflecting its peripheral position within the bank network. The Edisto population from the WTNWA Carolinian ecoregion, was distinct from all others, implicating the Florida Peninsula as a phylogeographic barrier. Biophysical particle-tracking simulations independently recovered the same spatial structure, with modeled larval exchange consistent with the genomic signal across the bank network. These findings reveal that S. exserta persists across the northern WTNWA province through a dual reproductive strategy of clonality at the bank scale and episodic larval exchange at the network scale, with implications for the conservation and management of mesophotic octocoral metacommunities.

ecology

From Prompt to Provenance: BloClaw, a Capability-Gated AI4S Workstation for Auditable Computational Biology

Scientific agents can produce plausible answers while remaining unable to establish whether the computation behind an answer is executable, recoverable, or reproducible. We present BloClaw, an AI4S workstation built around a simple principle: a scientific agent should know what it can do, show how it did it, and state what remains unvalidated. Each capability declares an execution state, input constraints, dependencies, expected outputs, and scientific limitations. Natural-language requests are translated into structured tasks, validated against this registry, executed through scientific tools, and recorded in a provenance-aware Living Lab Notebook. The system is designed to detect invalid inputs, failed tool calls, missing dependencies, and remote timeouts, and to route them to repair, retry, or escalation. The implemented and tested scope comprises RDKit-based molecular property and rule screening, protein structure analysis, docking-pose inspection, 3D visualization, and structured reporting. We demonstrate the workflow on a PubChem-retrieved osimertinib structure and a supplied 6LU7 docking artifact: the former yields deterministic descriptors (molecular weight 499.619 Da, cLogP 4.5098, TPSA 87.55 A^2), while the latter contains 2,387 protein ATOM records, 309 residues, and nine pose records. These examples are workflow demonstrations, not efficacy or affinity studies. Beyond retrospective prediction, the manuscript specifies a prior-minimized constructive mode in which a desired function is compiled into explicit physical, chemical, and systems constraints, candidate mechanisms are simulated, and observations are reintroduced for calibration and falsification; this is a proposed extension rather than a result of the present case studies. We describe an evaluation protocol that compares BloClaw with a standard single-agent workflow and fixed-script execution using task completion, scientific correctness, recovery success, provenance completeness, reproducibility, human review time, latency, and cost. This manuscript reports the system design, verified capability boundary, deterministic software artifacts, and a reproducible evaluation protocol; it does not claim benchmark improvements before those experiments are run. BloClaw is an execution and accountability layer for AI-assisted research, complementing expert review and experimental validation rather than replacing them.

bioinformatics

High-Resolution Subtyping of Pediatric Low-Grade Glioma Using an Integrated Meta-Clustering Framework

Pediatric low-grade glioma (pLGG) is the most common type of brain tumor in children, accounting for approximately 30% of all central nervous system tumors in children. pLGG has multiple molecular subtypes that differ in disease progression, recurrence patterns, and treatment responses. Conventional wet lab approaches including molecular profiling and histopathological studies for pLGG characterization are time consuming, costly, and laborious. Recently, methods based on artificial intelligence (AI) or machine learning (ML) have been widely used for pLGG molecular categorization, but most of them can only identify two or three pLGG subtypes. To more comprehensively characterize the molecular subtypes of pLGG and their potential biological and therapeutic significance, we develop an integrated meta-clustering approach, namely Meta-pLGG, that can explore high resolution molecular subtypes and their transcriptional heterogeneity for pLGG. Specifically, we first performed multiple rounds of random projection (RP) to generate dimension-reduced feature vectors from pLGG transcriptomics data, each of which was subsequently clustered by different clustering algorithms including hierarchical clustering, K-means, Self-Organizing Maps (SOM), Non-negative Matrix Factorization (NMF), Gaussian Mixture Model (GMM), and Spectral Clustering, as base clustering methods. Then, to yield robust clustering performance, we integrated the clustering results of these RP based individual clustering algorithms by adopting a weighted meta-clustering (wMetaC) approach. Results based on 532 pLGG patients suggested that our proposed approach demonstrated superior stability and discriminative powers for higher resolution pLGG subtyping compared to conventional approaches. Based on consensus matrix analysis, we identified two major pLGG mega-subtypes, with one further subdivided into three subgroups and the other into two. Then, we performed cluster specific differential gene expression analysis, molecular pathway analysis, and gene-drug-disease association analysis. The results showed that the identified five subgroups exhibited significant subtype-specific transcriptomic heterogeneity. In summary, our meta-clustering approach demonstrated much higher performance and robustness in identifying higher resolution molecular subtypes of pLGG, revealing the molecular heterogeneity within pLGG and potentially providing new insights for more precise molecular subtyping and precision therapy.

bioinformatics

Sympathetic activation and the force-frequency relationship in heart failure with reduced ejection fraction

Background Limited heart rate rise contributes to reduced exercise tolerance for people who have heart failure with reduced ejection fraction (HFrEF), yet rate-adaptive pacing does not improve functional capacity due to an attenuated force-frequency relationship (FFR). How the FFR relates to total peripheral resistance and sympathetic tone in HFrEF is unknown. Methods In a prospective, observational study, participants with HFrEF and controls underwent an incremental pacing protocol, during which heart rate was increased from 50 to 140 beats per minute. At each heart rate increment LV contractility was measured by echocardiography to determine the FFR, as well as continuous beat-to-beat measurement of systolic and diastolic blood pressures with a plethysmography device to determine cardiac output, total peripheral resistance and blood pressure variability (BPV). A microneurography study was then conducted to measure muscle sympathetic nerve activity (MSNA) during incremental pacing. Results A total of 157 participants with HFrEF and 55 controls (mean age 71.1{+/-}1.4 years, 172 (81.1%) male) underwent the pacing protocol. We observed single units in seven of 11 participants who participated in the microneurography study. In both groups, LV contractility and cardiac output increased until the peak of the FFR, after which these declined. We observed a reduction in total peripheral resistance, blood pressure variability, MSNA frequency and incidence coinciding with the peak of the FFR, beyond which these increased. Whilst these relationships were present in both groups, they were more evident in participants with HFrEF. Conclusions For people with HFrEF there is a bidirectional relationship between heart rate and sympathetic activation, with a nadir of sympathetic tone occurring at the peak of the FFR. Both excessively low and high heart rates are accompanied by greater sympathetic activation. Taken together, these data suggest that optimal heart rate targets for HFrEF are likely to be individual.

physiology

Beyond benchmark accuracy: machine-learning turnover-number predictors require system-level validation

Enzyme turnover numbers (kcat) are essential for kinetic models and enzyme-constrained genome-scale metabolic models (ecGEMs), but measured values are sparse and therefore increasingly estimated using machine learning (ML). Although these predictors are commonly evaluated by global regression metrics, their practical utility depends on how errors propagate through downstream models. We benchmarked six current kcat predictors on a curated BRENDA-derived dataset and five of them on EnzyExtract. To assess the influence of training-set proximity, we compared each benchmark dataset with the available training data for each predictor. We then used the predicted kcat values to parameterize ecGEMs of Saccharomyces cerevisiae and evaluated growth predictions across 19 conditions. We find that benchmark accuracy is moderate even on the BRENDA-derived dataset and drops sharply on EnzyExtract, where all predictors achieve R2 values of 0.20 or lower. This decline is accompanied by substantially lower overlap between the benchmark and training datasets, with exact sequence matches ranging from 24% to 78% for BRENDA, compared with 9% to 26% for EnzyExtract. However, that overlap alone does not explain differences in generalization across predictors. Moreover, downstream performance is also not explained by benchmark ranking. Across 19 conditions, none of the tool-specific ecGEMs consistently reproduces the experimentally observed variation in growth. In glucose minimal medium, the weakest benchmark performer yields the most accurate growth prediction in the downstream ecGEMs, whereas higher-ranked predictors produce larger deviations in growth. We trace this mismatch to localized errors at high-leverage positions in yeast's metabolic network, where underpredicted mitochondrial ADP/ATP carrier turnover numbers restrict adenine nucleotide exchange and impose an apparent limitation on cytosolic ATP supply. Relaxing this constraint shifts predicted growth toward the experimental reference. Thus, ML-derived kcat values can affect not only quantitative growth predictions but also the phenotype a mechanistic model appears to identify. These results argue for application-driven validation of biological parameter predictors in the downstream systems they are intended to support.

bioinformatics

Burning down the mouse: Effects of wildfire and post-fire reseeding on Sin Nombre virus prevalence in its reservoir host

Worldwide, physical habitats and biological communities are being reshaped by wildfire. Such changes have obvious potential to alter pathogen ecology, yet few studies, outside of those focused on ectoparasites, have tested the effects of wildfire on pathogen prevalence in wildlife. Even fewer have focused on the impacts of strategies for post-fire remediation on pathogen circulation. Here, we investigated the effect of wildfire and wildfire mitigation on the prevalence and viral load of Sin Nombre virus (SNV) infection in its reservoir host, the western deer mouse, using a study design of matched burned, unburned, and post-burn reseeded sites in northern New Mexico. In total, we screened 411 individual deer mice for SNV via RT-qPCR and analyzed the effect of wildfire and wildfire mitigation on relative mouse abundance, individual infection probability, site-level prevalence and viral load. Relative abundance was highest at reseeded sites, and model selection indicated that habitat structure, particularly the gradient from closed canopy to open-herbaceous habitat, was consistently associated with increased relative abundance. Similarly, SNV prevalence was significantly higher in reseeded sites than either burned or unburned sites but did not differ in burned versus unburned sites. Viral load did not differ between burn history types, and zero-inflated gamma hurdle models did not identify any strong predictors of viral load. Serendipitously, we were also able to investigate the impacts of an El Nino Southern Oscillation (ENSO) cycle on patterns of infection in a subsample of sites that were studied during and one year after an ENSO year and found that SNV prevalence increased significantly post-ENSO. Both reseeding and ENSO deliver resource pulses that can support increases in mouse density and thereby enhance SNV transmission. The impacts of post-fire management practices on SNV prevalence in its reservoir host that were revealed in this study should be considered when implementing such strategies and when utilizing treated areas.

ecology

Single nucleus RNA sequencing and spatial transcriptomics reveal unique functionalities of gray matter versus white matter oligodendrocytes in aging and Alzheimer's Disease

Oligodendrocyte (OL) dysfunction and white-matter (WM) vulnerability are increasingly recognized as important aspects of aging and Alzheimer's Disease (AD), yet human WM-focused, cellular-resolution transcriptomic data remain limited. Here, we profiled prefrontal WM from 48 brain donors spanning young adulthood and late-life with low versus high AD Neuropathologic Change (ADNC) using single-nucleus RNA sequencing followed by spatial transcriptomics (CosMx) in a subset of matched donors. We integrated aged WM OLs with a reference dorsolateral prefrontal cortex grey-matter (GM) OL dataset (SEA-AD) to define region- and pathology-associated OL programs. Across modalities, GM OLs exhibited a robust synapse/neurotransmitter-associated transcriptional signature relative to WM OLs, whereas this program was reduced with aging and attenuated in high ADNC GM. In contrast, WM OLs showed stronger immune-associated programs with aging and further enhancement in high ADNC, including cytokine/chemokine signaling and antigen presentation-related pathways. High ADNC WM OLs also displayed amplified proteostasis and stress-adaptation signatures, including selective upregulation of chaperone/heat shock genes and ferritin subunits, consistent with increased protein-folding demand and altered iron handling. To resolve OL state organization beyond static differential expression, we annotated OL sub-states using marker panels and inferred pseudotime-guided directional state-to-state flows within each tissue/condition stratum. This analysis identified a conserved newly formed differentiating (NFOL)/differentiating [->] lipid remodeling (APOE/ABCA1/LPL+) [->] Stress/ISR-reactive architecture, with a pronounced expansion of the Stress/ISR-reactive compartment and altered transition-associated pathway enrichment in high ADNC WM. Together, these data define WM-specific OL programs linked to aging and ADNC and nominate a stress/immune-enriched OL state landscape consistent with a putative senescence-like phenotype in diseased WM.

neuroscience

Breeding cassava for intercropping with cowpea: monoculture selection captures most intercrop selection gain, but targeted testing remains necessary

Intercropping dominates smallholder cassava production in sub-Saharan Africa, yet cassava breeding programs evaluate genotypes exclusively under monoculture. Despite consistently reported system-level yield advantages, cassava yield is reduced by 17 to 51% under intercropping, indicating a need to reduce this competitive disadvantage through breeding. However, the quantitative-genetic foundations of intercrop breeding remain uncharacterized for tropical root crop systems. We hypothesized that monoculture selection would capture most, but not all, genetic merit for intercropping and that a limited tester set would be sufficient if general mixing ability predominated. We evaluated 120 cassava clones previously selected under monoculture in IITA advanced yield trials, testing them under monoculture and in intercrop with two contrasting cowpea varieties across two years at Ibadan, Nigeria. Spatial mixed models quantified genetic variation, genotype by cropping system interaction, cross system genetic relationships, realized selection gain, mixing ability, tester effects, and land equivalent ratio. Intercropping reduced cassava fresh root yield by 19%, but total land equivalent ratios exceeded 1.0 for all clones, confirming a system-level land use advantage. Cassava performance under intercropping was heritable, with estimates of 0.50 to 0.75, and genotype by cropping system interaction was not significant. Genetic correlations between monoculture and intercrop performance were high and approached unity (rg = 0.92 to 0.99), and selection efficiency was 26 to 44% at 10% intensity, confirming that high genetic correlation does not guarantee effective indirect selection. Monoculture selection captured approximately two-thirds of direct intercrop gain. General mixing ability dominated, specific mixing ability was negligible, and producer effects explained 20 to 47% of intercrop variance. The two architecturally and phenologically contrasting cowpea varieties had limited influence on cassava rankings. Here, we show for the first time that cassava breeding for intercropping can retain monoculture selection during early stages while adding two representative cowpea testers at the advanced trial stage. This staged strategy aligns cassava breeding with diversified smallholder systems without creating a separate pipeline.

plant biology