The hyperinflammatory syndromes in critically ill patients, including trauma, sepsis, and acute lung injury, are characterized by dysregulated neutrophil responses that contribute to tissue damage and poor outcomes. Using murine models of cytokine storm induced by trauma and lung injury, we identified transforming growth factor β (TGFβ) as a central regulator of immune checkpoint in neutrophils. TGFβ signaling modulates neutrophil activation and upregulates the expression of programmed death-ligand 1 (PDL1). Disruption of TGFβ signaling during hyperinflammation restores the migratory capacity of neutrophils but leads to excessive activation, severe pulmonary tissue damage, and increased susceptibility to spontaneous bacterial infection in the lung. Mechanistically, PDL1 expression alters neutrophil behavior within lung capillaries, promoting intravascular clustering and restricting tissue infiltration. Targeted deletion of PDL1 in neutrophils reverses hyperinflammation-induced clustering, restores effective trafficking to infectious foci, and enhances host-protective immune function while limiting pathological neutrophil hyperactivation. These findings define a TGFβ-PDL1 regulatory axis that restrains the pathogenicity of neutrophils during hyperinflammation, revealing a checkpoint mechanism that balances host defense and tissue integrity.

Abstract Despite accelerating interest in using human induced pluripotent stem cell (hiPSC)–derived products for disease modeling and therapeutic development, there is substantial evidence that conventional culture approaches do not fully recapitulate natural embryonic nor lineage-committed states. It remains poorly understood how in vitro environmental conditions cause divergence from natural developmental trajectories, and current strategies emphasize restricted characterization of phenotype without appreciating the complexity of biology in maintaining pluripotency and driving differentiation. To address this knowledge gap, we examined hiPSC cell state during short-term culture in stirred-suspension bioprocesses under varying oxygen and agitation conditions. We profiled intracellular metabolic, transcriptional, and proteomic changes to characterize cellular responses to engineered environments and implications for cell phenotype. Using a random forest framework, we modeled population dynamics over time across metabolic and transcriptional programs and mapped those predictions onto hallmark biological signatures. This integrative approach captures and identifies environmentally reinforced programs, offering a framework to guide optimization of pluripotent cell state maintenance and differentiation.

ABSTRACT Toxoplasmosis, a disease caused by apicomplexan Toxoplasma gondii (Tg), is associated with various neuropsychiatric and behavioral conditions. Toxoplasmosis can cause serious complications for those with weakened immune systems and during pregnancy. Cathelicidins, peptides with antimicrobial and immunomodulatory functions, are critical factors in host defense against microbes, but their role in parasitic infections is less well understood. This study demonstrates the protective function of endogenous cathelicidin against hepatic damage caused by Tg infection in an oral infection model. We challenged wild-type (Camp+/+) and cathelicidin-deficient (Camp-/-) mice with low-virulent Type II strain of Tg (ME-49) cysts. Our findings demonstrate that Camp-/- mice exhibited more severe clinical manifestations, higher mortality rates, and more pronounced hepatic damage compared to their Camp+/+ counterparts. Histological liver examinations indicated significant necrotic hepatitis in Camp-/- mice, correlating with increased local concentrations of pro-inflammatory cytokines and proteomic upregulation of poly(ADP-ribose) polymerase 3 and guanylate-binding proteins. Increased cerebral inflammation and Tg cystogenesis were also observed in Camp-/- mice. Systemically, Camp-/- mice presented elevated levels of pro-inflammatory mediators, specifically interferon-gamma (Ifn-γ) and tumor necrosis factor-alpha (Tnf-α). In cultured macrophages, endogenous cathelicidin increased after Tg challenge, while Camp-/- bone marrow-derived macrophages released higher amounts of Tnf-α than their counterparts. We conclude that cathelicidin protects against liver injury and systemic deterioration induced by Tg infection by downregulating the synthesis of pro-inflammatory cytokines.

Serum-free expanded hair follicle mesenchymal stem cells (hfMSCs) maintained mesenchymal phenotype and multipotency during static and stirred suspension bioreactor (SSB) expansion and promoted histological cartilage repair in a murine full-thickness cartilage defect model, with minimal direct engraftment yet expressing of a number of secreted protein (in vitro and in vivo), supporting a primarily paracrine mechanism of action. Osteoarthritis is characterized by irreversible cartilage degeneration and limited intrinsic repair, and current treatments largely address symptoms rather than restoring cartilage structure. Mesenchymal stem cell therapies show promise for cartilage repair but are constrained by invasive tissue harvest, donor variability, and the need for scalable, serum-free biomanufacturing approaches. hfMSCs obtained by minimally invasive hair plucking represent an accessible MSC source, but their performance after serum-free static and SSB expansion for cartilage repair has not been fully defined. hfMSCs from 4 adult donors were adapted from fetal bovine serum-containing media into a fully defined serum-free medium (PPRF-MSC-6) and expanded in either 2D static flasks or SSBs using Cultispher G microcarriers under serum-free conditions. Phenotype and potency were assessed by flow cytometry for canonical MSC markers and lack of hematopoietic markers, along with tri-lineage (osteogenic, adipogenic, chondrogenic) differentiation assays and quantitative mass spectrometry-based proteomics with pathway analysis. A NOD SCID murine full-thickness femoral groove cartilage defect model was used; 1 wk postinjury, mice received intra-articular injections of 1 × 10⁵ hfMSCs (static- or SSB-expanded, male or female donor) or phosphate-buffered saline, followed by Safranin O-based histological scoring and immunofluorescent detection of human nuclear antigen and human SOX9 at 4 wk postinjury. Serum-free hfMSC expansion achieved robust proliferation in static culture (∼8- to 12-fold over 5 days) and comparable or greater proliferation in SSBs (∼15-fold in a representative donor), while preserving MSC surface marker expression and tri-lineage differentiation capacity across donors and culture formats. Both static- and SSB-expanded hfMSCs significantly improved histological cartilage repair scores relative to phosphate-buffered saline controls, with regenerated tissue showing Safranin O-positive matrix, restoration of cartilage architecture, and integration with native cartilage. Human cell engraftment within the repair tissue was low with minimal SOX9 co-localization, and proteomic profiling revealed only modest differences between static and SSB conditions, with ZNF703 as the sole protein significantly upregulated. hfMSCs expressed a number of secreted proteins in vitro and in vivo including follistatin and α2-Macroglobulin. hfMSCs derived from plucked hair follicles can be expanded under fully defined serum-free conditions in either static flasks or SSBs while retaining mesenchymal phenotype, multipotency, and cartilage reparative function in vivo. The comparable repair outcomes between static- and SSB-expanded cells, together with minimal proteomic alterations, indicate that scalable serum-free bioreactor processing can generate therapeutically competent hfMSCs suitable for translational cartilage repair applications. Potential donor- and sex-associated variability in proliferation, engraftment patterns, and repair scores highlight the need for donor screening and standardized potency assays, and future studies in more chronic and immunocompetent models are warranted to define long-term efficacy and mechanistic contributions of hfMSC paracrine signaling.

BACKGROUND:To date, we have lacked an understanding of how coronary artery disease (CAD) affects the extracellular vesicle (EV) profile of human pericardial fluid (PF) and there is a paucity of data querying whether PF-derived EVs have functional benefits. This study characterizes PF-derived EVs and assesses their impact on angiogenesis in vitro and in vivo. METHODS:PF was collected from patients with and without CAD. PF-derived EVs of different sizes were isolated and characterized using microfluidic resistive pulse sensing. Human coronary artery endothelial cells (HCAECs) were exposed to EVs. Uptake of EVs by HCAECs and their impact on cell proliferation was evaluated. HCAECs were analyzed for their migratory and angiogenic properties. The in vivo effects of PF-derived EVs were assessed using murine ischemia models. To elucidate putative mechanisms, proteomic analysis was performed followed by in silico pathway analysis and functional validation. RESULTS:Small and medium-sized EVs (sEV and mEV, respectively) were isolated from PF. HCAECs exposed to sEVs isolated from CAD patients exhibited a significant increase in proliferations, wound closure, and tube formation. Injection of sEVs isolated from the PF of patients with CAD into pericardial cavity of MI mice reduced cardiac fibrosis and improved cardiac function. Differential protein expression implicated key pathways in angiogenesis, apoptosis mitigation, and fibrosis as protective effects of PF-sEVs. CONCLUSION:We identify that PF-derived EVs exert cardioprotective effects by promoting angiogenesis and reducing fibrosis. Future studies should evaluate whether our findings can be recapitulated in a large animal model and a pilot clinical trial.

Sex differences in knee osteoarthritis (KOA) are well documented, but the molecular drivers within meniscal tissue remain unclear. We studied whether simulated microgravity (SMG) models sex‑dependent meniscal degeneration and whether the lipid scavenger receptor CD36 mediates these effects. Human menisci from total knee arthroplasty (TKA) donors were analyzed (histology n = 5 females/5 males; flow cytometry and RT‑qPCR n = 7 females/7 males). In mice, we assessed spontaneous anterior‑horn mineralization by micro‑CT at 4, 12, and 24 weeks in wild‑type (WT) and CD36‑deficient (Cd36−/−) animals. Four‑week menisci were then encapsulated in 1

Drug-resistant epilepsy affects one-third of patients; the ketogenic diet offers a metabolic alternative, but poor tolerability limits adherence. Ketone esters provide an alternative, though the optimal route is unclear. Prefrontal cortex proteomics was profiled in Kcna1-/- mice, a model of human temporal lobe epilepsy, treated with KE by either oral gavage or subcutaneous injection. Oral gavage predominantly altered proteins associated with synaptic vesicle trafficking, postsynaptic organization, and neurotransmitter transport, a profile consistent with enhanced synaptic plasticity and neuronal signaling. Subcutaneous injection induced widespread downregulation of proteins involved in synaptic structure, mitochondrial translation, and ubiquitination consistent with bioenergetic remodelling and proteostasis.

The pericardium plays an important homeostatic role for the neighboring heart, providing both lubrication and structural support. In vivo models have further identified a protective role for the pericardium in modulating cardiac remodeling following myocardial infarction, possibly through the actions of tissue-resident pericardial macrophages. Using patient-derived pericardial samples, we establish that human pericardial immune cells directly inhibit cardiac fibroblast fibrotic activity, and this action is dampened following myocardial infarction. Using single-cell RNA sequencing of patient pericardial fluid cells, we identify two pericardial macrophage subsets that are uniquely altered in response to myocardial infarction, which contributes to a shift in their effector molecule expression profiles. We confirm that fibronectin-expressing human pericardial macrophages are the primary driver of the pericardial antifibrotic actions through the release of cystatin C. Finally, we establish cystatin C as a myeloid cell-derived cardioprotective effector molecule in an in vivo model of myocardial infarction. Collectively, we uncover a molecular mechanism of the local immune environment that regulates cardiac remodeling after myocardial infarction.

BACKGROUND:Thyroid eye disease (TED) is a complex autoimmune disorder that is frequently disfiguring and sight-threatening. Recent advances have shifted treatment from surgery toward targeted biologic therapies like teprotumumab (TMB), an IGF-1R inhibitor that improves proptosis, diplopia, and inflammation. However, ocular surface symptoms often persist. METHODS:We conducted a longitudinal pre-post comparative study of patients with active, moderate-to-severe TED treated with TMB at a tertiary academic medical center, with tear sampling at two prespecified time points: baseline (collected within the 3 weeks strictly antecedent to the first infusion) and post-treatment (within 6 months of the final infusion). Adults with active, moderate-to-severe TED who received ≥4 TMB infusions were eligible. All patients contributed paired pre- and post-treatment tear samples (22 eyes). Controls were adults without TED or ocular surface disease (32 eyes). Tear fluid was collected by Schirmer strips and analyzed by liquid chromatography-tandem mass spectrometry. Differential expression was assessed using a linear mixed-effects model to explicitly account for the intrinsic correlation between paired eyes; correlations between LFQ intensities and clinical metrics used Spearman rank correlation. RESULTS:A total of 2974 proteins were identified across 76 tear samples. Unsupervised analyses demonstrated separation between control, pre-treatment, and post-treatment TED tear proteomes. Although TMB induced substantial proteomic changes, post-treatment profiles did not revert to a control-like state. Proteins involved in inflammation, oxidative stress, and cytoskeletal organization remained persistently dysregulated, while a subset of tear film stability-associated proteins normalized. Several persistently dysregulated protein targets overlapped with FDA-approved drugs. CONCLUSIONS:These observational findings support tear proteomics as a platform for biomarker discovery and therapeutic prioritization and warrant validation in larger, independent cohorts.

Osteoarthritis (OA) is a painful, debilitating disease with no cure or treatments that can predictably stop/reverse its progression. Treatment is particularly difficult since articular cartilage lacks intrinsic repair capacity, despite mesenchymal stem cells (MSCs) being present in the joint with robust chondrogenic potential. While heterogeneity exists among MSC subtypes within human synovium, it remains unclear which populations can regenerate cartilage or impact OA progression. We clonally isolated MSCs from normal and OA patient synovium using indexed flow cytometry, then characterized them through differentiation assays and quantitative proteomics. MSC clones were transplanted into a xenograft rat OA model and evaluated by histology and immunofluorescence. We identified heterogeneity in putative MSCs within and between patient groups and their repair capacity in the rat model. However, traditional cell surface markers could not distinguish these subtypes, highlighting the need for single-cell level understanding. Using unbiased proteomics, we identified CD47 as a novel MSC marker. CD47Hi cells demonstrated robust treatment efficacy in the rat OA model and directly contributed to new articular cartilage formation. Characterizing MSC subtypes is essential for identifying candidates appropriate for clinical investigation and exploiting functional MSCs for cartilage regeneration strategies.

Brain metastasis occurs in up to 40% of patients with non-small cell lung cancer (NSCLC). Considerable genomic heterogeneity exists between the primary lung tumor and respective brain metastasis; however, the identity of the genes capable of driving brain metastasis is incompletely understood. Here, we carried out an in vivo genome-wide CRISPR activation (CRISPRa) screen to identify molecular drivers of brain metastasis from an orthotopic NSCLC patient-derived xenograft model. We discovered activating expression of the Alzheimer’s disease associated β-site amyloid precursor protein cleaving enzyme 1 (BACE1) led to a significant increase in brain metastasis. Furthermore, genetic and pharmacological inhibition of BACE1 blocked NSCLC brain metastasis. Mechanistically, we identified BACE1 acts through its novel substrate EGFR to drive this metastatic phenotype. Together, our data highlights the power of in vivo CRISPR screening to unveil novel molecular drivers and potential therapeutic targets of NSCLC brain metastasis.

Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, which has an important role in developmental processes. Loss of Capn15 in mice leads to developmental eye anomalies and volumetric changes in the brain. Human individuals with biallelic variants in CAPN15 have developmental delay, neurodevelopmental disorders, as well as congenital malformations, including eye anomalies. However, the substrates of Capn15 are still unidentified. Here, using Capn15 KO P2 mice of both sexes, we have used RNA sequencing (RNA-SEQ), proteomics, and N-terminomics/terminal amino isotopic labelling of substrates (TAILS), to examine putative substrates of Capn15. There were few changes in the transcriptome profile, and we could not verify a protein change in one selected mRNA between Capn15-/- and WT mice, although a putative transcription factor linked to these changes, Pax2, did show a significant increase after the loss of Capn15. TAILS revealed a preference for cleavage at basic residues, and while no hits showed a significant change in cleavage, some were more abundant when Capn15 was removed. These included Doublecortin and Tubb3, and the Doublecortin predicted cleavage was at a lysine residue. Cleavages at lysine residues were enriched in peptides that were lost or reduced when Capn15 was removed, but not in cleavages that were unchanged when Capn15 was removed.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high mortality and limited therapeutic options. Microglia and macrophages are rapidly recruited to the lesion site and contribute substantially to secondary brain injury. However, the key molecular mediators that drive their neurotoxic effects remain incompletely understood. We investigated the role of extracellular matrix metalloproteinase inducer (EMMPRIN, also known as CD147) in promoting microglia/macrophage-mediated neurotoxicity after ICH. EMMPRIN was selectively deleted in myeloid cells using both AAV-mediated knockdown and CX3CR1Cre:EMMPRINfl/fl mice. Neuronal survival and functional outcomes were assessed using histological, molecular, and behavioral analyses. Targeted deletion of EMMPRIN in microglia/macrophages significantly reduced neuronal death and improved neurological recovery following ICH. Mechanistically, EMMPRIN-mediated neurotoxicity was associated with elevated expression of matrix metalloproteinases and enhanced activation of the p38 mitogen-activated protein kinase (MAPK) pathway, and with downstream engagement of myocyte enhancer factor 2 C (MEF2C) and B-cell lymphoma 2 (Bcl2). Notably, EMMPRIN deletion also enhanced neurogenesis and oligodendrogenesis in the perihematomal region, suggesting a potential role in promoting endogenous brain repair. These findings establish EMMPRIN elevation in myeloid cells as a prominent regulator of ICH pathophysiology and a promising therapeutic target to limit secondary injury and promote brain repair.

Aberrant levels of the cysteine protease Calpain-2 have been linked to neurodegeneration, inflammation, and cancer, yet our understanding of this protease and its substrates remains limited. Systematic studies to identify Calpain-2 substrates have been largely confined to peptide libraries or in vitro studies, which fail to represent physiological cellular conditions and physiologically relevant substrates. To identify existing and novel Calpain-2 substrates, we used a genetic approach to knockout Calpain-2 in the THP-1 human monocyte-like cells, followed by proteomic and N-terminomic/TAILS mass spectrometry approaches to identify Calpain-2 substrates. We identified 51 substrates that may be cleaved directly by Calpain-2 or indirectly by downstream proteases. The direct cleavage of selected substrates by Calpain-2 was confirmed using in vitro assays. Finally, metabolomics analysis identified a role for Calpain-2 in the regulation of pyrimidine and glutathione metabolism. Our unbiased and quantitative mass spectrometry analytical pipeline provides new evidence on the physiological functions of Calpain-2 and its newly identified substrates in THP-1 cells.

A hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most prevalent genetic cause of ALS, with early neuromuscular junction (NMJ) dysfunction being a key pathological feature. Current therapies provide only limited symptomatic relief, underscoring the need for targeted, mechanism-based interventions. Using a C9orf72 ALS zebrafish model (C9-miR) and patient-derived induced pluripotent stem cell (iPSC) motor neurons, we identified significant downregulation of calpastatin, the endogenous inhibitor of calpains, a calcium-dependent protease family implicated in neurodegeneration. We demonstrate that restoring calpastatin function through calpain inhibition with calpeptin or a novel calpastatin-derived peptide ameliorates locomotor deficits and NMJ dysfunction in the C9-miR zebrafish model. These interventions enhance synaptic vesicle turnover and quantal release at the NMJ while improving motor neuron excitability and synaptic integrity in iPSC-derived motor neurons. N-terminomic/TAILS mass spectrometry approaches revealed direct calpain-mediated cleavage of synaptic proteins in motor neurons derived from C9orf72 patients. Proteolysis of key synaptic proteins, such as spectrin, is prevented by calpeptin and calpastatin peptide treatments. Our findings establish the calpastatin/calpain axis as a pivotal regulator of synaptic function in C9orf72-associated ALS and identify it as a promising therapeutic target, offering a novel strategy to restore synaptic transmission and potentially halt disease progression.

Pulmonary microvascular endothelial cell (PMVEC) intercellular junctions are critical for maintaining barrier function and mitigating pulmonary edema. Previously, we demonstrated that aging exacerbated pulmonary microvascular permeability in a model of lung injury. Based on this, we hypothesized that aging was associated with increased PMVEC barrier dysfunction due to impaired cell–cell junction integrity. PMVEC were isolated from young and aged mice and cultured to confluence in vitro. Barrier function, junctional integrity, alterations in the proteome, markers of inflammation, and actin cytoskeleton organization were all assessed. To model injurious conditions, PMVEC were stimulated with inflammatory cytokines. PMVEC from aged mice exhibited increased permeability, both under basal and inflammatory conditions, which was associated with disrupted cell‐surface localization of the adherens junction protein, vascular endothelial (VE)‐cadherin. Protein abundance of VE‐cadherin was increased with age, while levels of the adapter protein, ‐catenin, and the tight junction protein, claudin‐5, were decreased. Measures of inflammation, including cytokine expression and cell surface abundance of adhesion molecules, did not differ with age. Augmented presence of actin stress fibers was observed in aged PMVEC. We conclude that aging predisposes PMVEC to elevated injury, due to inherent deficiencies in cell–cell junctions and barrier function, potentially mediated through altered actin cytoskeleton organization.

Infantile epileptic spasms syndrome (IESS) is a rare, early-onset pediatric epilepsy with severe neurological consequences. In refractory cases, the ketogenic diet (KD) is used as a metabolic therapy. To understand the antiepileptic mechanisms of the diet, this study examined the proteomic profiles in the hippocampus after KD treatment. Utilizing a rodent model of IESS in combination with quantitative proteomics, this study showed that the KD upregulated proteins involved in synaptogenesis, mitochondrial function, and neuroinflammation. Preliminary investigation reveals the KD induces alterations in hippocampal protein expression that mitigate neurodevelopmental consequences, providing candidate targets for future IESS investigation.

Lung injury leads to pulmonary microvascular endothelial cell (PMVEC) damage, disruption of cell-cell junctions, and increased permeability. Previously, we demonstrated in a mechanical ventilation-induced model of lung injury that aging exacerbated pulmonary microvascular permeability. Based on this, we hypothesized that aging was associated with increased PMVEC barrier dysfunction due to impaired cell-cell junction integrity. PMVEC were isolated from young and aged mice and cultured to confluence in vitro . Barrier function and cell-cell junction integrity were assessed through electric cell-substrate impedance sensing, XPerT permeability assay, immunofluorescence, and western blot analysis. Further studies were conducted to examine alterations in the proteome, markers of inflammation, and actin cytoskeleton organization. To model injurious conditions, PMVEC were stimulated with inflammatory cytokines; permeability and actin cytoskeletal alterations were subsequently assessed. We observed increased basal permeability in PMVEC from aged mice, which was associated with disrupted cell-surface localization of the adherens junction protein, vascular endothelial (VE)-cadherin. Protein abundance of VE-cadherin was significantly increased with age; however, levels of the adapter protein, γ -catenin, and the tight junction protein, claudin-5, were decreased. Measures of inflammation, including cytokine expression and cell surface abundance of adhesion molecules, did not differ with age. Alterations in actin cytoskeleton organization, characterized by augmented presence of actin stress fibers, were observed in aged PMVEC. Under inflammatory conditions, permeability and actin stress fiber formation were exacerbated with age. It is concluded that aging predisposes PMVEC to elevated injury, due to inherent deficiencies in cell-cell junctions and barrier function, potentially mediated through altered actin cytoskeleton organization. New and Noteworthy Compared with pulmonary microvascular endothelial cells (PMVEC) from young mice, PMVEC isolated from aged mice had higher permeability, which was directly associated with impairments in cell-cell junctions. The higher permeability in aged PMVEC was not associated with augmented inflammatory signaling but was associated with actin cytoskeletal alterations. Following an inflammatory insult, PMVEC from aged mice had further exacerbated permeability. These findings may begin to highlight why older patients exhibit higher mortality during lung injury. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institutes of Health Research, https://ror.org/01gavpb45 London Health Sciences Centre Research Institute Western University, https://ror.org/02grkyz14 Ontario Graduate Scholarship Dean’s Research Scholarship

MicroRNAs (miRNAs) are essential regulators involved in multiple biological processes. To achieve their gene repression function, they are loaded in miRNA-specific Argonautes to form the miRNA-induced silencing complex (miRISC). Mammals and C. elegans possess more than one paralog of miRNA-specific Argonautes, but the dynamic between them remains unclear. Here, we report the conserved dipeptidyl peptidase DPF-3 as an interactor of the miRNA-specific Argonaute ALG-1 in C. elegans. Knockout of dpf-3 increases ALG-2 levels and miRISC formation in alg-1 loss-of-function animals, thereby compensating for ALG-1 loss and rescuing miRNA-related defects observed. DPF-3 can cleave an ALG-2 N-terminal peptide in vitro but does not appear to rely on this catalytic activity to regulate ALG-2 in vivo. This study uncovers the importance of DPF-3 in the miRNA pathway and provides insights into how multiple miRNA Argonautes contribute to achieving proper miRNA-mediated gene regulation in animals.