European Journal of Cell Biology

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

The methyltransferase EZH2 (Enhancer of Zest Homolog 2) and the demethylase KDM6B (Lysine Demethylase 6B) have been associated with epithelial to mesenchymal transition (EMT) and poor prognosis in various cancers. These enzymes methylate and demethylate H3K27me3 and regulate distinct sets of genes controlling EMT induction, despite having opposite catalytic activities. This could be due to their recruitment or the modulation of their activity by partner proteins on specific loci. This work sought to identify proteins associated with chromatin and interacting with EZH2 or with KDM6B during EMT. To do so, co-immunoprecipitation and mass spectroscopy was used under TGF{beta} (Tumor growth factor {beta}) and TNF (Tumor necrosis factor ) treatment to induce EMT in A549 lung cancer cells. Surprisingly, numerous proteins related to focal adhesions were identified to interact with EZH2 or KDM6B. These proteins are part of a nuclear protein interaction network previously described as nucleo-adhesome. Among these proteins, TGFB1I1 (transforming growth factor induced peptide 1) and CSRP2 (cysteine and glycine rich protein 2) were further confirmed to interact with KDM6B in the nucleus and even more so during EMT. The target genes of these complexes were then sought by knocking down KDM6B, TGFB1I1 or CSRP2. Three genes (coding Integrin alpha 5, Laminin y2 and Matrix Metalloproteinase 9) were confirmed to be regulated by KDM6B, TGFB1I1 and CSRP2. These findings may have clinical relevance, as immunohistochemistry analyses performed on a cohort of lung cancer patients revealed increased nuclear localization of TGFB1I1 and CSRP2 in cells undergoing EMT.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

Injuries to individual cells happen frequently as a result of physiological and environmental stresses during their normal daily functions that can lead to a ruptured cell cortex (plasma membrane and underlying cortical cytoskeleton). The capacity of cells to rapidly repair general daily injuries, as well as ones resulting from trauma, infection, or diseases/cancer, is essential for their survival. While we know the general cell biological outline of the highly-conserved physiological events taking place during cell wound repair, our knowledge of the molecular mechanisms governing the repair process is still fairly limited, due in large part to the lack of information regarding the molecules, machineries, and pathways involved. Here, we performed a genetic screen of 1322 fluorescent-tagged proteins to identify cell wound repair components that are recruited upon laser wounding or whose expression is lost and/or altered upon laser wounding. We identified 129 proteins that are recruited to wounds during the cell repair process through high resolution spatio-temporal expression analyses of these gene fusions in conjunction with a fluorescent actin reporter. Strikingly, we find that many members of the Rab family GTPases are recruited to wounds where, in addition to their well-known roles in intracellular membrane trafficking, they are affecting actin cytoskeletal organization and dynamics during the repair process. These studies are allowing us to define the earliest acting proteins, as well as those required at specific steps in the repair process based on their recruitment patterns and the precise timing of their recruitment to wounds. Thus, our imaging-based screen is providing us with a global view of the repair processes, as well as a large number of genes/gene families that provide new entry points for examining specific steps in the cell wound repair process. Author SummaryCells in our bodies get injured every day from normal activity, environmental stress, infection, or disease. To survive, they must quickly repair these injuries and restore normal function. While some molecules have been identified as key players of cell wound repair, many of the molecules involved and their roles remain unknown. In this study, we identified new molecules that are involved in different steps of cell wound repair. Using laser-induced injury in the Drosophila model, we examined 1322 proteins and observed their spatial and temporal dynamics in a cell after injury. From the 1322 proteins examined, we identified 129 proteins recruited to distinct regions around the damage site during cell wound repair, suggesting roles in specific steps of the repair process. Interestingly, a subset of these proteins are Rab family GTPase members, highlighting new roles for these proteins in regulating actin dynamics. By identifying new candidate repair molecules, we provide a foundation for understanding how cells maintain their integrity and how repair processes may be influenced by factors such as wound size, infection, aging, and disease.

Endothelial-to-mesenchymal transition (EndMT) has become a central mechanism in developmental biology, fibrosis, vascular disease, and cancer. We previously reported on an integrated signaling model in which TGF-{beta}2 induces EndMT through coordinated activation of Smad-dependent and Smad-independent signaling pathways converging on Snail, while GSK-3{beta} regulates Snail activity. We performed a systematic figure-by-figure reproducibility analysis of the original publication. Independent studies published between 2011 and 2026 were identified and curated according to predefined inclusion criteria. Each original experimental conclusion was evaluated for independent confirmation. In parallel, selected biochemical experiments were independently reproduced using newly acquired reagents and contemporary Western blot methodologies. Independent publications consistently reproduced each major mechanistic conclusion of the original study, including activation of Smad, ERK, PI3K/AKT, and p38 MAPK signaling, regulation of Snail expression, EndMT-associated marker switching, and GSK-3{beta}-dependent control of Snail activity. Independent laboratory experiments reproduced the principal biochemical findings using contemporary reagents and experimental workflows. The combined literature analysis and independent laboratory replication demonstrate that the mechanistic framework in our previous study has remained reproducible across multiple laboratories, endothelial cell types, disease models, and fifteen years of investigation. This work illustrates a complementary framework for assessing reproducibility that integrates direct experimental replication with cumulative independent validation.

Adult zebrafish efficiently regenerate skeletal muscle following different types of injury; however, the molecular programs involved in repair after extensive cryoinjury remain to be comprehensively characterized. Here, we explored the transcriptomic response of adult zebrafish skeletal muscle at 7 days post cryoinjury (dpci), a stage marked by ongoing tissue clearance, progenitor expansion, myogenic differentiation, and nascent myofiber formation, and compared it with phase-matched stab wound injury. Cryoinjury induced a broad transcriptional response, with 5,330 differentially expressed genes. Integrated enrichment and protein-protein interaction analyses revealed that, at 7 dpci, zebrafish skeletal muscle functions as an integrated regenerative system in which immune remodeling, progenitor expansion, myogenic differentiation, extracellular matrix reconstruction, mechanotransduction, biosynthetic adaptation, proteostasis, and intracellular trafficking operate simultaneously. In parallel, mature sarcomeric and oxidative metabolic programs were suppressed, consistent with ongoing tissue reconstruction and structural immaturity. Comparison with stab-wounded skeletal muscle revealed substantial transcriptional conservation, as 612 of 717 stab-wound-responsive genes (85%) were also differentially expressed after cryoinjury. Shared upregulated genes formed coherent functional modules related to proliferation, extracellular matrix organization and signaling, immune regulation, muscle differentiation, and protein processing. Thus, distinct injury modalities converge on a common regenerative program, while cryoinjury elicits a quantitatively broader transcriptional response. These findings support a conserved regenerative architecture of adult zebrafish skeletal muscle repair, in which interconnected biological modules act coordinately, with the breadth of transcriptional engagement reflecting regenerative demand.

BackgroundLamins are major regulators of the spatial and functional organization of chromatin. Lamins at the nuclear periphery form the lamina that anchors heterochromatin to the nuclear envelope. A subpool of A-type lamins localizes in the nuclear interior, where they also bind to euchromatic genomic regions. A-type lamin properties and chromatin association are regulated by lamin-associated polypeptide 2alpha (LAP2). Here we systematically analyze, how LAP2 depletion affects chromatin organization, accessibility and gene expression on a genome-wide level. ResultsLAP2 depletion in mouse dermal fibroblasts positively and negatively affects chromatin accessibility and gene expression throughout the genome, which correlates with changes in chromatin association of A-type lamins and the nucleosomal remodeler proteins BRG1 and CHD4. In particular, A-type lamins bind to open chromatin regions close to BRG1 and CHD4 binding sites and deregulated genes, but do not directly accumulate on genes and BRG1 and CHD4-enriched sites. Unsupervised clustering of the datasets on LAP2-bound genomic regions confirms spreading of A-type lamins to active chromatin regions containing deregulated genes and an enrichment of chromatin remodelers on a subset of these genomic regions. ConclusionsLAP2 depletion in fibroblasts leads to a gross rearrangement of chromatin. Genome-wide chromatin reorganization is linked to spreading of A-type lamins to active chromatin regions and accompanied by a restriction of chromatin remodelers to a subset of active genomic regions. These changes correlate with changes in chromatin accessibility and gene expression throughout the genome, particularly in regions where lamin binding is gained in LAP2 knockout versus wildtype cells.

Biomolecular condensates are membraneless compartments critical for the functional organization of cellular macromolecules in essential processes such as cell division, gene transcription or stress responses. We previously reported that vimentin filaments remodel into phase separated biomolecular condensates upon oxidative stress. This process requires vimentin single cysteine, C328, suggesting the involvement of oxidative modifications of this residue. Here, we aimed to generate vimentin condensates by inserting mutations mimicking posttranslational modifications associated with oxidative stress. In vimentin deficient cells, a cysteine oxidation mimetic mutant, vimentin C328D, formed only elongated particles or short filaments that evolved towards droplets upon serum deprivation or treatment with the oxidant diamide. Among vimentin posttranslational modifications rapidly responding to these stimuli, glycosylation confers filament stability whereas phosphorylation promotes disassembly. We observed that the O-deglycosylation inhibitor thiamet G, and the kinase inhibitors staurosporine and H-89, attenuated diamide-elicited vimentin C328D droplet formation, suggesting a potential glycosylation/phosphorylation interplay in this effect. Indeed, introducing phosphomimetic residues at certain single vimentin glycosylation and/or phosphorylation sites induced the formation of droplets, only if combined with the C328D mutation. In particular, the vimentin S49D,C328D mutant formed condensates that were reversibly dispersed by dilution through hypotonic shock. Therefore, mimicking C328 oxidation and S49 phosphorylation was sufficient to elicit vimentin phase separation. In vitro, purified vimentin S49D,C328D polymerized into a mixture of aberrant filaments and aggregates, which, in the presence of crowders, evolved towards paracrystals or clusters of beaded assemblies depending on pH. These findings highlight the role of C328 perturbations in the formation of biomolecular condensates and suggest a modulatory role of glycosylation/phosphorylation, thus shedding light on the processes regulating vimentin phase separation.

The mechanical properties of the nucleus are critical for maintaining nuclear integrity and function. We previously showed that chromatin dominates short-extension mechanics whereas lamins provide long-extension strain stiffening. To distinguish the roles of lamin isoforms, micromanipulation nucleus force measurements were performed on isolated nuclei from lamin A/C (Lmna-/-) and lamin B1 (Lmnb1-/-) knockout mouse embryonic fibroblast cells. Lamin A/C knockout does not alter short-extension nuclear stiffness but is essential for strain stiffening at longer extensions. Oppositely, lamin B1 loss reduced short-extension stiffness due to facultative heterochromatin loss while long-extension strain stiffening was slightly increased. Loss of lamin A/C and B1 resulted in similar lamin-chromatin linkers effects as LBR did not change and LAP2{beta} decreased in both. A simulation model of a polymeric lamin shell with stiff lamin A/C and softer lamin B1 subunits can qualitatively recapitulate experimental measurements of lamin knockout cells. Lamin A/C knockout resulted in abnormal nuclear shape but not nuclear blebbing or rupture whereas lamin B1 knockout, similar to other perturbations that cause heterochromatin loss, resulted in increased nuclear blebbing and rupture. This work illuminates the distinct mechanical roles of lamin A/C and B1 in determining nuclear structure and integrity.

Nitrite can be reduced to nitric oxide (NO) by several heme- and molybdenum-containing proteins, including mitochondrial cytochrome c oxidase (Cco). This activity, designated Cco/NO, has been implicated in hypoxic signaling, but its regulation and quantitative significance relative to other NO-producing systems remain uncertain. We examined its modulation by adenine nucleotides using detergent-solubilized yeast and mouse brain mitochondria supplied with 1 mM nitrite and an ascorbate/TMPD/cytochrome c electron-donor system. ADP and ATP differentially modulated Cco/NO activity, and ADP extended measurable NO formation across the entire oxygen range tested, up to the assay ceiling of 175 {micro}M O2. Nucleotide regulation was also isoform-dependent: ATP slightly inhibited Va-containing Cco but strongly stimulated Vb-containing Cco under anoxic conditions. Rates normalized to cytochrome aa demonstrate multi-turnover nitrite-reductase capacity under these substrate-driven assay conditions. Both the cellular ADP/ATP ratio and subsequently assayed Cco/NO activity increased transiently following a hypoxic shift. These findings establish metabolic and isoform-dependent gating of the catalytic capacity of Cco/NO; they do not establish its fractional contribution to total cellular NO or its operation at physiological nitrite concentrations in intact, coupled mitochondria. This research was supported by CONICET Grant PIP 706 (research team member P.R.C.) and National Institutes of Health Grant GM30228 to R.O.P.

Immune checkpoint blockade has revolutionized cancer therapy, but the therapeutic efficacy is limited. Clinical trials on blockade of newly identified immune checkpoints didn't show promising result, suggesting that it might be insufficient to understand the function of immune checkpoints in cancer merely in the context of immunity. Here, we found mutually exclusive expression patterns of the immune checkpoint VISTA (or VSIR) and the neural stemness factor SETDB1, an oncoprotein that promotes immunoevasion, in xenograft tumors, suggesting that cells with high VISTA expression represents a differentiated, and hence, less or non-malignant state in tumor. Non-neural differentiation factors HHEX, MYOD1 and PPARG promote, whereas oncoproteins KRAS (and the mutant KRAS(G12D)) and SOX2, both being embryonic neural factors, repress VISTA expression. This tendency can be inferred from the finding that neural stemness is the core property of cancer cell. Manipulated expression of VISTA in cancer cells generated no significant effect on cell tumorigenicity and differentiation state, but led to change in cell morphology and actin cytoskeleton. Mechanistically, VISTA regulates a key cytoskeleton regulator, WASF2, leading to the change in cell morphology, which might interfere with signal transduction of immune response. The results suggest that 1) high expression of a protein in tumor might represent a less or non-malignant state, targeting of which would leave malignant cells intact, and consequently, leading to weak or even no therapeutic efficacy, a key factor worth considering for target selection; 2) immune checkpoints might play other roles in cells that interfere with regulation of anti-tumor immunity.

Chondrosarcoma is a hyper-vascularised, chemoresistant cartilage malignancy driven by VEGF-centred angiogenesis, and local adipose depots are increasingly recognised as paracrine drivers of tumour angiogenesis via adipokines and extracellular vesicles (EVs). The infrapatellar fat pad (IFP), an inflammatory adipose depot within the articular joint in direct cartilage contact, is a key local source of adipose-derived EVs, and thus a candidate driver of angiogenesis in chondrosarcoma. The aim of this study was to determine whether the IFP is a productive source of EVs, and whether IFP-derived EVs induce angiogenesis in articular chondrocytes. The IFP released significantly more EVs than subcutaneous fat (n = 8 per depot; p = 0.027). Treating primary human articular chondrocytes with IFP EVs for 24 h upregulated VEGFA (+1.6-fold, p = 0.036) and downregulated BMP4 (-2.4-fold, p = 0.011), engaging the VEGF/eNOS/ERK axis that drives chondrosarcoma angiogenesis. Re-analysis of a previously published phospho-kinase dataset from the same donor EVs, corroborated by a pooled donor-group analysis (n = 3), supported activation of eNOS, ERK1/2, PLC-{gamma}1 and HSP27. These findings identify the IFP as a dominant source of EVs within the articular joint, which can induce a pro-angiogenic, VEGF-axis switch in articular cartilage cells, supporting a signalling model relevant to chondrosarcoma angiogenesis.

At the onset of intervertebral disc degeneration, within the disc core, the pH and osmolarity decrease, and the residing notochordal cells (NCs) gradually transition towards nucleus pulposus cells (NPCs). How these microenvironmental cues shape the NCs extracellular vesicles (EV)-enriched secretome, and thus EV-mediated communication with NPCs during this transition, remains poorly understood. To study this, we collected the secretome from pig NC-rich tissue cultured for 4 days in either healthy or degenerate disc media to mimic these changes. In both conditions, NC-rich tissues were largely comparable at the histological and biochemical levels. Despite, tissues released glycosaminoglycans (GAGs), depleting the extracellular matrix. Surprisingly, degenerative media did not differentially release inflammatory regulators, though it reduced PGE2 release. We asked whether this extended to EV-enriched secretome media (SM_EV+), and found that the degenerative media reduced the number of EVs without altering their morphology or size. We then determined NC-EV association of inflammatory and matrix regulators. NC-EV isolation enriched MMP1, IL6 and IL10 and depleted soluble GAGs. Conversely, EV-depletion (SM_EV-) removed most GAGs without affecting MMP1, IL6, and IL10, suggesting that they contribute to the NC-EV soft corona. Functionally, healthy SM_EV+ improved GAG production by NPCs, but attenuated TBXT expression. Degenerate SM_EV+ did not elicit detectable EV-specific effects. These findings suggest that, in health, secretome-mediated communication from NCs to NPCs is only partially EV-mediated. At the onset of IVD degeneration, low pH and osmolarity impair the release of NC-EVs and negate the EV-specific beneficial matrix-anabolic effects on NPCs, contributing to the NC-to-NPC transition.

Background: Lumbar disc herniation, with or without concomitant disc degeneration, is a major cause of lumbar radiculopathy and low back pain, which also a key public musculoskeletal disorder without an exact pathophysiology. Studies have suggested that inflammatory cells and biochemical markers of inflammation also play an important role in lumbar radiculopathy in addition to nerve compression. The aim of the present study was to assess the association of selected circulatory inflammatory markers (CRP, hs-CRP and E-selectin) in patients with lumbar disc herniation without radiological degeneration (LDH) and lumbar disc herniation with radiological degeneration (LDHD). Materials & methods: This case-control study included 208 participants, comprising 104 patients with lumbar disc pathology and 104 controls. Patients were further stratified into LDH (n=67) and LDHD (n=37). Serum CRP, hs-CRP and E-selectin concentrations were measured. Results: Among the patients, 35.6 % presented with LDHD while 64.4 % had only LDH. Significantly increased median hs-CRP (p<0.001) and CRP (p<0.001) were observed in patients groups compared to controls, while CRP showing a consistent independent association across the combined disease (OR=1.68, 95% CI=1.33-2.14, p<0.001), LDHD (OR=1.62, 95% CI=1.16-2.20, p=0.005) and LDH (OR=1.69, 95% CI=1.30-2.20, p<0.001) multivariable models. No significant difference was observed in serum E-selectin between the study groups. Multivariable models incorporating inflammatory and clinical variables demonstrated substantially greater discriminatory performance than individual biomarkers alone. Conclusion: Elevated circulating CRP and hs-CRP concentrations were associated with lumbar disc pathology, with CRP showing a consistent independent association across the combined disease, LDH and LDHD multivariable models, whereas E-selectin showed no significant association. Multivariable models incorporating inflammatory and clinical variables demonstrated greater discriminatory performance than individual biomarkers. These findings support a potential systemic inflammatory component in lumbar disc pathology, although the cross-sectional nature of the measurements does not establish causality or a local inflammatory response within the disc.

Orthodontic tooth movement relies on a tightly regulated pro-inflammatory and pro resorptive mechanoresponse of local periodontal ligament fibroblasts (PdLFs). Dysregulation is linked to complications such as root resorption and tooth loss. Hyperlipidemic conditions promote excessive PdL mechanoresponses, with growth differentiation factor 15 (GDF15) acting as potential regulator. This study examined the contribution of the inflammasome/pyroptosis pathway as underlying mechanism for dysregulated mechanoresponses. Human PdLFs were treated with palmitic acid (PA) or oleic acid (OA) for six days before 24 hours of compressive loading. PA increased CASP1, CASP4, and CASP3 activity, secretion of IL-1{beta}, IL-18, and HMGB1, and LDH release. Pharmacological blockade and siRNA-mediated knockdown of inflammasome- and pyroptosis-related targets revealed that NLRP3, CASP1, CASP4, and GSDMD partially contributed to monocyte and osteoclast overactivation. Silencing PA-increased GDF15, partially normalized the phenotype, at least in part by inflammasome/pyroptosis regulation. GDF15 acted through extracellular, and a nuclear signaling route, each accounting partially to this phenotype. Together, GDF15 partially regulates the PA-induced, pyroptosis-associated overactivated mechanoresponse alongside pyroptosis-independent mechanisms suggesting it as an interesting target for potential clinical interventions.

Matrix metalloproteinase 13 (MMP13) is a zinc-dependent protease that plays key roles in extracellular matrix remodeling. Like several other MMPs, MMP13 has been shown to interact with heparan sulfate (HS), a highly sulfated glycosaminoglycan found at the cell surface and in the extracellular matrix, but the significance of the interaction remains unknown. Here we report that while zymogen and mature forms of MMP13 both bind HS with high affinity, their interactions with HS display markedly different characteristics in terms of preferred HS structure and binding kinetics. By structure-guided mutagenesis, we identified a large HS-binding site of MMP13 consists of 10 residues in the hemopexin domain, 3 residues in the catalytic domain, and 2 residues in the linker region. While these basic residues participate in binding to both zymogen and mature forms of MMP13, the relative contribution of many residues differs substantially between the two forms, which likely contributes to their distinct HS-binding characteristics. Binding of HS to mature MMP13 resulted in selective inhibition of the collagenase activity of MMP13 in a length- and sulfation-dependent manner, but the binding had no effect on degradation of non-collagen substrates. Mechanistically, the inhibitory effect of HS likely results from reduced interdomain flexibility after binding of HS, and/or HS-induced dimerization of MMP13. In sum, our study establishes HS as a multifaceted regulator of MMP13 activity, and discovers that the HS-binding site of MMP13 is a novel exosite that can be targeted to inhibits its collagenase activity.

The perilipins generally represent the most abundant lipid droplet (LD) surface proteins in mammalian cells and can target LD subpopulations within the same cell. They are characterized by a conserved organization of disordered and folded regions, as well as a number of divergent features, which contribute to differences in perilipin function and LD targeting. Here, we focus on the C-terminal 4-helix bundle (4HB) domain that is present in all perilipins except for PLIN1. Using biochemical and in silico approaches, we show that the 4HB of PLIN3 is a stably folded domain and interacts with lipid surfaces in vitro and with LDs in model cells. The {beta}-subdomain at the bottom of the helical bundle is required for the binding to LDs, but not for the 4HB stability, suggesting that this region may promote direct interaction with the LD surface. In agreement, the 4HB of PLIN4, which does not contain an {beta}- subdomain, does not bind to LDs. Overall, our work shows that small differences in perilipin structural features impact their differential targeting to LDs.

Background: Newly available arm angle data offers a new dimension to understand rising rates of arm injury in MLB pitchers. Purpose: To evaluate the relationship between arm angle, pitch characteristics, and elbow and forearm injury in MLB pitchers.<br><br> Study Design: Retrospective cohort study; Level of evidence, 3 Methods: Statcast data from 2020 to 2025 and MLB injured list (IL) data were used to evaluate arm angle and pitch characteristics in relation to elbow and forearm injuries. Results are presented with and without requirements on prior season workload and for same-season and next-season injury incidence. A generalized additive model (GAM) was used to capture non-linear dependence and interactions between selected features and injury incidence to the elbow or forearm. Average marginal effect (AME) odds ratios are reported for main effect terms. Results: N = 3,812 pitcher-seasons were included. 29% pitchers who underwent UCLR did so in the same season as a forearm injury (tmean=44, tmedian=27 days to surgery). Arm angle, fastball usage, and their interaction were the three most predictive features. Arm angle was positively related to incidence of injury (ORmeanAME=1.014), fastball usage was inversely related to incidence of injury (ORmeanAME=0.243), and arm angle moderated the effect fastball usage at high arm angle, where increased usage was no longer protective. Slider velocity (ORmeanAME=1.072), spin rate (ORmeanAME=1.001), and usage (ORmeanAME=2.039) also significantly predicted injury risk. Fastball velocity was not significant in any fit, with ORmeanAME=0.999 across all fits. Fit-level Nagelkerke R2 values ranged from .019 to .052. Conclusion: Fastball usage and arm angle, not velocity, predicted elbow and forearm injury risk among MLB pitchers, and arm angle was the single most predictive feature. The heterogeneity of risk factors as a function of arm angle, and the novelty of MLB arm angle data, may explain why fastball usage has been previously underexplored as a risk factor. Keywords: baseball; arm angle; fastball velocity; fastball usage; spin rate; UCL; ulnar collateral ligament; elbow injury; forearm injury; Statcast

Traumatic optic neuropathy (TON) occurs due to direct or indirect injury to the optic nerve and is a significant cause of visual disability. So far, there is no effective treatment. The lack of understanding of the cellular mechanisms by which trauma induces inflammation and damage in retinal neurons is a critical knowledge gap in developing effective therapies. We have studied the role of the arginase 1 (A1) enzyme in this pathology. We have found previously that treatment with a long-acting form of human recombinant A1, pegylated A1 (PEG-A1) after optic nerve crush limits activation of retinal microglia and macrophages (M{Phi}) and reduces inflammation, thereby decreasing injury and protecting visual function. Here we report on studies designed to demonstrate the therapeutic efficacy of PEG-A1 in mouse models of direct and indirect TON and to elucidate the underlying mechanisms. We used ONC to model direct TON and sonication-induced trauma to the supraorbital rim to model indirect TON (SI-TON). At different times after injury, mice were treated with PEG-A1 which was delivered systemically by i.p. injection or locally by intravitreal injection. In order to assess the role of A1-induced activation of the ornithine/polyamine pathway in the protective effects of PEG-A1, some mice were treated with the ornithine decarboxylase (ODC) inhibitor, difluoromethylornithine (DFMO) immediately after the PEG-A1 treatment. Retinal function was determined by OptoMotry and electroretinography. Retinal injury and microglia/M{Phi} activation were assessed by immunofluorescence imaging. Expression of inflammatory cytokines was determined by Western blotting and quantitative RT PCR. Liquid chromatography mass spectrometry was used to analyze changes in arginase/ODC pathway metabolites. Results showed that PEG-A1 treatment improved neuronal survival and visual function whether delivered systemically or intravitreally. This neuroprotection was associated with decreased microglia/M{Phi} activation, decreased inflammatory cytokine expression, and increased formation of L-ornithine and putrescine. Furthermore, DFMO treatment blocked these effects, indicating that PEG-A1 limits retinal injury and preserves vision after ocular injury by activating ODC. ODC processes the arginase product L-ornithine to form polyamines which are known to promote reparative functions. Thus, PEG-A1 therapy offers a new strategy to limit trauma-induced vision loss and promote repair after TON.

Syndecans are transmembrane proteoglycans that regulate angiogenesis through both their glycosaminoglycan chains and core proteins. While roles for all four mammalian syndecans in new blood vessel formation are well established, it has more recently emerged that their extracellular core proteins contain discrete bioactive regulatory sequences capable of influencing cellular processes, including angiogenesis. We previously demonstrated that the syndecan-3 (SDC3) ectodomain possesses anti-angiogenic activity independent of its heparan sulphate chains. Here, we identified and characterised a novel anti-angiogenic sequence within the SDC3 ectodomain. Using recombinant truncation mutants, endothelial migration assays and peptide mapping, we localised activity to a discrete region of the extracellular domain and subsequently defined a conserved minimal nine amino acid peptide, QM111, that retained full biological activity. QM111 inhibited endothelial cell migration and angiogenic sprouting in both rat aortic ring and mouse choroidal explant models. Intrinsic disorder analysis revealed that QM111 resides within a region of comparatively reduced disorder, consistent with other syndecan regulatory sequences. This supports the concept that syndecan ectodomains contain conserved functional modules embedded within intrinsically disordered extracellular domains. QM111 did not induce inflammatory chemokine production, exhibited no detectable cytotoxicity, and retained substantial stability in human serum and vitreous humour. Finally, QM111 displayed anti-angiogenic activity comparable to the previously described syndecan-2-derived peptide QM107, with combination treatment producing more robust inhibition of angiogenesis. These findings identify QM111 as a novel endogenous anti-angiogenic peptide and support the concept that syndecan ectodomains are reservoirs of biologically active regulatory sequences with therapeutic potential. The work further establishes syndecan-derived peptides as a promising platform for the development of next-generation anti-angiogenic therapies.