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NLR family, apoptosis inhibitory proteins (NAIPs) detect bacterial flagellin and structurally related components of bacterial type III secretion systems (T3SS), and recruit NLR family CARD domain containing protein 4 (NLRC4) and caspase-1 into an inflammasome complex that induces pyroptosis. NAIP/NLRC4 inflammasome assembly is initiated by the binding of a single NAIP to its cognate ligand, but a subset of bacterial flagellins or T3SS structural proteins are thought to evade NAIP/NLRC4 inflammasome sensing by not binding to their cognate NAIPs. Unlike other inflammasome components such as NLRP3, AIM2, or some NAIPs, NLRC4 is constitutively present in resting macrophages and not known to be induced by inflammatory signals. Here, we demonstrate that Toll-like receptor (TLR)-dependent p38 mitogen-activated protein kinase signaling up-regulates NLRC4 transcription and protein expression in murine macrophages, which licenses NAIP detection of evasive ligands. In contrast, TLR priming in human macrophages did not up-regulate NLRC4 expression, and human macrophages remained unable to detect NAIP-evasive ligands even following priming. Critically, ectopic expression of either murine or human NLRC4 was sufficient to induce pyroptosis in response to immunoevasive NAIP ligands, indicating that increased levels of NLRC4 enable the NAIP/NLRC4 inflammasome to detect these normally evasive ligands. Altogether, our data reveal that TLR priming tunes the threshold for the murine NAIP/NLRC4 inflammasome to enable inflammasome responses against immunoevasive or suboptimal NAIP ligands. These findings provide insight into species-specific TLR regulation of NAIP/NLRC4 inflammasome activation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Purpose: Patients with locally advanced rectal cancer often require neoadjuvant chemoradiation therapy to downstage the disease, but the response is variable with no predictive biomarkers. We have previously revealed through proteomic profiling that myoferlin is associated with response to radiation therapy. The aims of this study were to further validate this finding and explore the potential for myoferlin to act as a prognostic and/or therapeutic target., Methods and Materials: Immunohistochemical analysis of a tissue microarray (TMA) for 111 patients was used to validate the initial proteomic findings. Manipulation of myoferlin was achieved using small interfering RNA, a small molecular inhibitor (wj460), and a CRISPR-Cas9 knockout cell line. Radiosensitization after treatment was assessed using 2-dimensional clonogenic assays, 3-dimensional spheroid models, and patient-derived organoids. Underlying mechanisms were investigated using electrophoresis, immunofluorescence, and immunoblotting., Results: Analysis of both the diagnostic biopsy and tumor resection samples confirmed that low myoferlin expression correlated with a good response to neoadjuvant long-course chemoradiation therapy. High myoferlin expression was associated with spread to local lymph nodes and worse 5-year survival (P = .01; hazard ratio, 3.5; 95% CI, 1.27-10.04). This was externally validated using the Stratification in Colorectal Cancer database. Quantification of myoferlin using immunoblotting in immortalized colorectal cancer cell lines and organoids demonstrated that high myoferlin expression was associated with increased radioresistance. Biological and pharmacologic manipulation of myoferlin resulted in significantly increased radiosensitivity across all cell lines in 2-dimensional and 3-dimensional models. After irradiation, myoferlin knockdown cells had a significantly impaired ability to repair DNA double-strand breaks. This appeared to be mediated via nonhomologous end-joining., Conclusions: We have confirmed that high expression of myoferlin in rectal cancer is associated with poor response to neoadjuvant therapy and worse long-term survival. Furthermore, the manipulation of myoferlin led to increased radiosensitivity in vitro. This suggests that myoferlin could be targeted to enhance the sensitivity of patients with rectal cancer to radiation therapy, and further work is required., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: To utilize machine learning for identifying treatment response genes in diabetic foot ulcers (DFU)., Methods: Transcriptome data from patients with DFU were collected and subjected to comprehensive analysis. Initially, differential expression analysis was conducted to identify genes with significant changes in expression levels between DFU patients and healthy controls. Following this, enrichment analyses were performed to uncover biological pathways and processes associated with these differentially expressed genes. Machine learning algorithms, including feature selection and classification techniques, were then applied to the data to pinpoint key genes that play crucial roles in the pathogenesis of DFU. An independent transcriptome dataset was used to validate the key genes identified in our study. Further analysis of single-cell datasets was conducted to investigate changes in key genes at the single-cell level., Results: Through this integrated approach, SCUBE1 and RNF103-CHMP3 were identified as key genes significantly associated with DFU. SCUBE1 was found to be involved in immune regulation, playing a role in the body's response to inflammation and infection, which are common in DFU. RNF103-CHMP3 was linked to extracellular interactions, suggesting its involvement in cellular communication and tissue repair mechanisms essential for wound healing. The reliability of our analysis results was confirmed in the independent transcriptome dataset. Additionally, the expression of SCUBE1 and RNF103-CHMP3 was examined in single-cell transcriptome data, showing that these genes were significantly downregulated in the cured DFU patient group, particularly in NK cells and macrophages., Conclusion: The identification of SCUBE1 and RNF103-CHMP3 as potential biomarkers for DFU marks a significant step forward in understanding the molecular basis of the disease. These genes offer new directions for both diagnosis and treatment, with the potential for developing targeted therapies that could enhance patient outcomes. This study underscores the value of integrating computational methods with biological data to uncover novel insights into complex diseases like DFU. Future research should focus on validating these findings in larger cohorts and exploring the therapeutic potential of targeting SCUBE1 and RNF103-CHMP3 in clinical settings., Competing Interests: Declarations Ethical approval and consent to participate This study did not involve any human participants or animals, and therefore ethical approval and consent to participate were not required. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The aberrant expression of placental imprinted genes due to epigenetic alterations during pregnancy can impact fetal development. We investigated the impact of dietary modification of low vitamin B12 with varying doses of folic acid on the epigenetic control of imprinted genes and fetal development using a transgenerational model of C57BL/6J mice. The animals were kept on four distinct dietary combinations based on low vitamin B12 levels and modulated folic acid, mated in the F0 generation within each group. In the F1 generation, each group of mice is split into two subgroups; the sustained group was kept on the same diet, while the transient group was fed a regular control diet. After mating, maternal placenta (F1) and fetal tissues (F2) were isolated on day 20 of gestation. We observed a generation-wise opposite promoter CpG methylation and gene expression trend of the two developmental genes Dlk1 and Grb10, with enhanced gene expression in both the sustained and transient experimental groups in F1 placentae. When fetal development characteristics and gene expression were correlated, there was a substantial negative association between placental weight and Dlk1 expression (r = - 0.49, p < 0.05) and between crown-rump length and Grb10 expression (r = - 0.501, p < 0.05) in fetuses of the F2 generation. Consistent with these results, we also found that H3K4me3 at the promoter level of these genes is negatively associated with all fetal growth parameters. Overall, our findings suggest that balancing vitamin B12 and folic acid levels is important for maintaining the transcriptional status of imprinted genes and fetal development., Competing Interests: Declarations Ethics approval and consent to participate All the animal experiments were conducted after approval from the Institutional Animal Ethics Committee, Post Graduate Institute of Medical Education and Research, Chandigarh (PGIMER/IAEC/566). Consent for publication All authors provided consent for publication. Competing interests The authors declare that there are no competing interests., (© 2024. The Author(s).)

Myoferlin (MYOF) is a muscle-derived secretory protein. Recent studies have found that MYOF protects against cell damage. However, the role of MYOF in cardiac hypertrophy remains unclear. Increasing evidence suggests that NLRP3 (NOD-like receptor protein 3) and the pyroptosis cascade play critical roles in the development of cardiac hypertrophy and inflammation. To investigate the role of MYOF in cardiac hypertrophy, we conducted a transverse aortic constriction (TAC) experiment in a mouse model. We found that MYOF can improve cardiac hypertrophy and cardiac function. Furthermore, our study confirmed a connection between cardiac hypertrophy and myocardial pyroptosis. Cardiac hypertrophy significantly increased the proportion of apoptotic cells and upregulated apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and gasdermin D (GSDMD). This suggests that pharmacological or genetic inhibition of NLRP3 can effectively reduce cardiac hypertrophy. An abnormal increase in NLRP3 can reverse the cardioprotective effects of MYOF. Our findings indicate that MYOF is a potential therapeutic agent for cardiac hypertrophy., Competing Interests: The authors declare there are no competing interests., (©2024 Zhou et al.)

Triple-negative breast cancer (TNBC) continues to pose a significant obstacle in the field of oncology. Dysregulation of lipid metabolism, notably upregulated ketogenesis, has emerged as a hallmark of TNBC, yet its role in metastasis has been elusive. Here, by utilizing clinical specimens and experimental models, the study demonstrates that increased ketogenesis fosters TNBC metastasis by promoting the up-regulation of β-hydroxybutyrate (β-OHB), a key ketone body. Mechanistically, β-OHB facilitates β-hydroxybutyrylation (Kbhb) of Calpastatin (CAST), an endogenous calpain (CAPN) inhibitor, at K43, blocking the interaction with CAPN and subsequently promoting FAK phosphorylation and epithelial‒mesenchymal transition (EMT). In conclusion, the study reveals a novel regulatory axis linking ketogenesis to TNBC metastasis, shedding light on the intricate interplay between metabolic reprogramming and tumor progression., Competing Interests: Declarations Ethical approval All experiments received approval from the Clinical Trial Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (KY2024-R080) and were conducted in accordance with the Helsinki Declaration principles. Animal experiments were conducted with the approval of the Animal Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University in accordance with the Regulations on the Management of Experimental Animals. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Phospholamban (PLN) plays a crucial role in regulating sarcoplasmic reticulum (SR) Ca 2+ cycling and cardiac contractility. Mutations within the PLN gene have been detected in patients with cardiomyopathy, with the heterozygous variant c.40&#95;42delAGA (p.R14del) of PLN being the most prevalent. Investigations into the mechanisms underlying the pathology of PLN-R14del have revealed that cardiac cells from affected patients exhibit pathological aggregates containing PLN. Herein, we performed comprehensive molecular and cellular analyses to delineate the molecular aberrations associated with the formation of these aggregates. We determined that PLN aggregates contain autophagic proteins, indicating inefficient degradation via the autophagy pathway. Our findings demonstrate that the expression of PLN-R14del results in diminished autophagic flux due to impaired fusion between autophagosomes and lysosomes. Mechanistically, this defect is linked to aberrant recruitment of key membrane fusion proteins to autophagosomes, which is mediated in part by changes in Ca 2+ homeostasis. Collectively, these results highlight a novel function of PLN-R14del in regulating autophagy, that may contribute to the formation of pathogenic aggregates in patients with cardiomyopathy. Prospective strategies tailored to ameliorate impaired autophagy may hold promise against PLN-R14del disease., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication All authors agree to the publication of this study. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. A.G.E is co-founder and CSO of GENOSOPHY, a spin-off company of the National and Kapodistrian University of Athens., (© 2024. The Author(s).)

Background: Vascular calcification is a major contributor to cardiovascular disease, especially diabetes, where it exacerbates morbidity and mortality. Although pyrophosphate is a recognized natural inhibitor of vascular calcification, there have been no prior studies examining its specific deficiency in diabetic conditions. This study is the first to analyze the direct link between elevated glucose levels and disruptions in extracellular pyrophosphate metabolism., Methods: Rat aortic smooth muscle cells, streptozotocin (STZ)-induced diabetic rats, and diabetic human aortic smooth muscle cells were used to assess the effects of elevated glucose levels on pyrophosphate metabolism and vascular calcification. The techniques used include extracellular pyrophosphate metabolism assays, thin-layer chromatography, phosphate-induced calcification assays, BrdU incorporation for DNA synthesis, aortic smooth muscle cell viability and proliferation assays, and quantitative PCR for enzyme expression analysis. Additionally, extracellular pyrophosphate metabolism was examined through the use of radiolabeled isotopes to track ATP and pyrophosphate transformations., Results: Elevated glucose led to a significant reduction in extracellular pyrophosphate across all diabetic models. This metabolic disruption was marked by notable downregulation of both the expression and activity of ectonucleotide pyrophosphatase/phosphodiesterase 1, a key enzyme that converts ATP to pyrophosphate. We also observed an upregulation of ectonucleoside triphosphate diphosphohydrolase 1, which preferentially hydrolyzes ATP to inorganic phosphate rather than pyrophosphate. Moreover, tissue-nonspecific alkaline phosphatase activity was markedly elevated across all diabetic models. This shift in enzyme activity significantly reduced the pyrophosphate/phosphate ratio. In addition, we noted a marked downregulation of matrix Gla protein, another inhibitor of vascular calcification. The impaired pyrophosphate metabolism was further corroborated by calcification experiments across all three diabetic models, which demonstrated an increased propensity for vascular calcification., Conclusions: This study demonstrated that diabetes-induced high glucose disrupts extracellular pyrophosphate metabolism, compromising its protective role against vascular calcification. These findings identify pyrophosphate deficiency as a potential mechanism in diabetic vascular calcification, highlighting a new therapeutic target. Strategies aimed at restoring or enhancing pyrophosphate levels may offer significant potential in mitigating cardiovascular complications in diabetic patients, meriting further investigation., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

An ideal cell surface target has ubiquitously high cancer expression, absence from healthy tissues, and an essential role cancer initiation and/or maintenance. In this issue of Cancer Cell, Hamilton et al. combine proteomics, transcriptomics, epigenomics, and dependency databases to identify DLK1, a novel immunotherapeutic target for neuroblastoma., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlates with a super-enhancer. Immunofluorescence, flow cytometry, and immunohistochemistry show robust cell surface expression of DLK1. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells results in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Since high DLK1 expression is found in several adult and pediatric cancers, our study demonstrates the utility of a proteogenomic approach and credentials DLK1 as an immunotherapeutic target., Competing Interests: Declaration of interests F. Zammarchi, K. Havenith, and P.H.v.B. are or were employed by ADC Therapeutics at the time the work was conducted and hold or previously held shares/stocks in ADC Therapeutics. The following patent is held: WO2018146199A1., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Angiogenesis is essential for repairing critical-sized bone defects. Although adipose-derived stem cell (ADSC)-derived exosomes have been shown to enhance the angiogenesis of endothelial progenitor cells (EPCs), the underlying mechanisms remain unclear. This study aims to explore the effects and mechanisms of ADSC-derived exosomes in enhancing bone repair by promoting EPC angiogenesis., Methods: Transmission electron microscopy, nanoparticle tracking analysis, and Dil reagent kit were employed to identify ADSC-derived exosomes and their internalization by EPCs. Micro-CT analysis, H&E staining, and Masson staining were used to assess bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), as well as the pathological changes and fibrosis at defect sites. Cell viability, migration, invasion, and tube formation of EPCs were evaluated using CCK-8, wound healing, Transwell, and tube formation assays. Immunohistochemical staining, RT-PCR, and Western blotting were utilized to measure the gene and protein expression of markers such as CD31, VEGFA, OCN, RUNX2, NOTCH1, and DLL4. Gene sequencing and bioinformatics analyses were conducted to identify the most highly expressed miRNA in exosomes, while miRDB and dual-luciferase reporter assays were used to explore the interaction between miR-21-5p and NOTCH1., Results: The ADSC-derived exosomes, averaging 126 nm in diameter, were internalized by EPCs. In vivo, these exosomes promoted new bone formation, increased BMD, BV/TV, Tb.Th, and Tb.N, reduced pathological damage to cranial defect tissues, enhanced vascular and bone tissue regeneration, and upregulated OCN and RUNX2 expression. In vitro, ADSC-derived exosomes enhanced EPC viability, migration, invasion, and tube formation. Both in vivo and in vitro experiments demonstrated that ADSC-derived exosomes upregulated CD31 and VEGFA expression. miR-21-5p, the most highly expressed miRNA in ADSC-derived exosomes, was found to target NOTCH1. Overexpression of miR-21-5p in these exosomes facilitated EPC migration, tube formation, and VEGFA expression while downregulating NOTCH1 and DLL4 expression. Inhibition of miR-21-5p produced opposite effects on EPCs., Conclusions: These findings indicate that miR-21-5p in ADSC-derived exosomes promotes angiogenesis in EPCs to accelerate bone repair by targeting the NOTCH1/DLL4/VEGFA signaling pathway, offering a potential therapeutic strategy for bone defect treatment., (© 2024. The Author(s).)

Cerebral ischemia disrupts calcium homeostasis in the brain causing excitotoxicity, oxidative stress, inflammation, and neuronal cell apoptosis. During ischemic conditions, T-type calcium channel channels contribute to increase in intracellular calcium ions in both neurons and glial cells therefore, the current study hypothesizes the antagonism of these channels using ML218, a novel specific T-Type inhibitor in experimental model of cerebral ischemia-reperfusion (CI/R) brain injury. CI/R injury was induced in Swiss Albino mice by occlusion of common carotid arteries followed by reperfusion. Animals were assessed for learning and memory (MWM), motor coordination (Rota rod), neurological function (neurological deficit score), cerebral infarction, edema, and histopathological alterations. Biochemical assessments were made for calcium binding proteins (Calmodulin- CaM, calcium/calmodulin-dependent protein kinase II-CaMKII, S100B), oxidative stress (4-hydroxy 2-nonenal-4-HNE, glutathione-GSH, inflammation (nuclear factor kappa-light-chain-enhancer of activated B-p65-NF-kB, tumor necrosis factor-TNF-α, interleukin-IL-10) inducible nitric oxide synthase (iNOS) levels, and acetylcholinesterase activity (AChE) in brain supernatants. Furthermore, serum levels of NF-kB, iNOS, and S100B were also assessed. CI/R animals showed impairment in learning, memory, motor coordination, and neurological function along with increase in cerebral infarction, edema, and histopathological alterations. Furthermore, increase in brain calcium binding proteins, oxidative stress, inflammation, and AChE activity along with serum NF-kB, iNOS, and S100B levels were recorded in CI/R animals. Administration of ML218 (5 mg/kg and 10 mg/kg; i.p.) was observed to recuperate CI/R induced impairments in behavioral, biochemical, and histopathological analysis. Hence, it may be concluded that ML218 mediates neuroprotection during CI/R via decreasing brain and serum calcium binding proteins, inflammation, iNOS, and oxidative stress markers., Competing Interests: Declaration of competing interest Authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aims: SCUBE2 (signal peptide-CUB-epidermal growth factor-like domain-containing protein 2) is a secreted or membrane-bound protein originally identified from endothelial cells (ECs). Our previous work showed that SCUBE2 forms a complex with E-cadherin and stabilizes epithelial adherens junctions (AJs) to promote epithelial phenotypes. However, it remains unclear whether SCUBE2 also interacts with vascular endothelial (VE)-cadherin and modulates EC barrier function. In this study, we investigated whether and how SCUBE2 in ECs regulates vascular barrier maintenance., Methods and Results: We showed that SCUBE2 colocalized and interacted with VE-cadherin and VE-protein tyrosine phosphatase (VE-PTP) within EC AJs. Furthermore, SCUBE2 knockdown disrupted EC AJs and increased EC permeability. Expression of EC SCUBE2 was suppressed at both mRNA and protein levels via the nuclear factor-κB signalling pathway in response to pro-inflammatory cytokines or permeability-inducing agents. In line with these findings, EC-specific deletion of Scube2 (EC-KO) in mice impaired baseline barrier function and worsened vascular leakiness of peripheral capillaries after local injection of histamine or vascular endothelial growth factor. EC-KO mice were also sensitive to pulmonary vascular hyperpermeability and leucocyte infiltration in response to acute endotoxin- or influenza virus-induced systemic inflammation. Meanwhile, EC-specific SCUBE2-overexpressing mice were protected from these effects. Molecular studies suggested that SCUBE2 acts as a scaffold molecule enabling VE-PTP to dephosphorylate VE-cadherin, which prevents VE-cadherin internalization and stabilizes EC AJs. As such, loss of SCUBE2 resulted in hyperphosphorylation of VE-cadherin at tyrosine 685, which led to its endocytosis, thus destabilizing EC AJs and reducing barrier function. All of these effects were exacerbated by inflammatory insults., Conclusion: We found that SCUBE2 contributes to vascular integrity by recruiting VE-PTP to dephosphorylate VE-cadherin and stabilize AJs, thereby promoting EC barrier function. Moreover, our data suggest that genetic overexpression or pharmacological up-regulation of SCUBE2 may help to prevent vascular leakage and oedema in inflammatory diseases., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) belongs to the SPARC family of matricellular proteins. However, underlying functions of SPARCL1 in bladder cancer (BCa) remain understudied. We performed an integrated search for the expression patterns of SPARCL1 in relation to various clinicopathological features of BCa. We then carried out Gene Ontology (GO) enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and gene set enrichment analysis (GSEA). Furthermore, we investigated the correlations between SPARCL1 and immunological features, such as tumour mutation burden (TMB), immune activation processes, immune checkpoint expression, tumour immune dysfunction and exclusion (TIDE) scores, and chemotherapeutic sensitivity in BCa. Our analysis revealed that SPARCL1 was downregulated across multiple cancers. In BCa, elevated SPARCL1 was linked with advanced histopathologic stage, higher T and N stage, and poorer prognosis in the clinical cohort. In vitro experiments demonstrated that increased SPARCL1 expression inhibited cell proliferation, migration, and invasion. Additionally, highly expressed SPARCL1 was linked to elevated immune, stromal and ESTIMATE scores, as well as an increase in naive B cells, M2 macrophages, and resting mast cells. We observed a moderate correlation between SPARCL1 expression and CD163, VSIG4 and MS4A4A, which are markers of M2 macrophages. Furthermore, SPARCL1 expression was positively related to TMB, immune activation processes, TIDE scores, immune checkpoint expression, and chemotherapeutic sensitivity in BCa. Our study highlights the potential involvement of SPARCL1 in macrophage recruitment and polarization and suggests its utility as a biomarker for prognosis in BCa., (© 2024 The Author(s). Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The calcium-binding protein spermatid-associated 1 (CABS1) localizes to the principal piece of mature sperm flagella. Deletion of CABS1 results in subfertility in male mice, possibly due to an impaired annulus in the sperm flagella. However, it is unknown whether there are other mechanisms by which CABS1 affects male fertility. Our current investigation has uncovered that CABS1 is located in the midsection of the flagellum in testicular sperm and the principal piece in epididymal sperm. Moreover, male mice lacking CABS1 exhibit a defect in the progressive motility of sperm. Furthermore, the regulation of calcium levels, which has been reported to have a significant impact on sperm motility, capacitation, and the acrosome reaction, is also affected when sperm are exposed to alkalized high-salt buffer (pH 8.0) and progesterone (100 μM) in Cabs1-null spermatozoa. This alteration in calcium response may contribute to changes in the phosphorylation of PKA substrates and subsequent phosphorylation of tyrosine residues. Additionally, the absence of CABS1 leads to a defective fibrous sheath and abnormal configuration of doublet microtubules in post-testicular sperm. These findings indicate that the absence of CABS1 also disrupts the structural integrity of the fibrous sheath, resulting in male subfertility. The highly conserved nature of CABS1 in humans suggests that it could potentially be a contributing factor to asthenozoospermia in men., (© 2024 Wiley Periodicals LLC.)

Calcineurin is a serine/threonine protein phosphatase that is highly conserved from yeast to human and plays a critical role in many physiological processes. Regulators of calcineurin (RCANs) are a family of endogenous calcineurin regulators, which are capable of inhibiting the catalytic activity of calcineurin in vivo and in vitro. In this study, we first characterized the biochemical properties of yeast calcineurin and its endogenous regulator Rcn1, a yeast homolog of RCAN1. Our data show that Rcn1 inhibits yeast calcineurin toward pNPP substrate with a noncompetitive mode; and Rcn1 binds cooperatively to yeast calcineurin through multiple low-affinity interactions at several docking regions. Next, we reinvestigated the mechanism underlying the inhibition of mammalian calcineurin by RCAN1 using a combination of biochemical, biophysical, and computational methods. In contrast to previous observations, RCAN1 noncompetitively inhibits calcineurin phosphatase activity toward both pNPP and phospho-RII peptide substrates by targeting the enzyme active site in part. Re-analysis of previously reported kinetic data reveals that the RCAN1 concentrations used were too low to distinguish between the inhibition mechanisms [Chan B et al. (2005) Proc Natl Acad Sci USA 102, 13075]. The results presented in this study provide new insights into the interaction between calcineurin and RCAN1/Rcn1., (© 2024 Federation of European Biochemical Societies.)

Regulation of mitochondrial calcium uptake by the mitochondrial calcium uniporter (mtCU) complex is crucial for heart function. In a recent study, Hasan et al. demonstrated that mitochondrial calcium uptake (MICU)1 and MICU2, regulatory subunits of the complex, help maintain calcium homeostasis in cardiac mitochondria, providing potential targets for therapies aimed at improving mitochondrial function in heart disease., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Inflammasome sensors activate cellular signaling machineries to drive inflammation and cell death processes. Inflammasomes also control the development of certain diseases independently of canonical functions. Here, we show that the inflammasome protein NLR family CARD domain-containing protein 4 (NLRC4) attenuated the development of tumors in the Apc min/+ mouse model. This response was independent of inflammasome signaling by NLRP3, NLRP6, NLR family apoptosis inhibitory proteins, absent in melanoma 2, apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1 and caspase-11. NLRC4 interacted with the DNA-damage-sensing ataxia telangiectasia and Rad3-related (ATR)-ATR-interacting protein (ATRIP)-Ewing tumor-associated antigen 1 (ETAA1) complex to promote the recruitment of the checkpoint adapter protein claspin, licensing the activation of the kinase checkpoint kinase-1 (CHK1). Genotoxicity-induced activation of the NLRC4-ATR-ATRIP-ETAA1 complex drove the tumor-suppressing DNA damage response and CHK1 activation, and further attenuated the accumulation of DNA damage. These findings demonstrate a noninflammatory function of an inflammasome protein in promoting the DNA damage response and mediating protection against cancer., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) play a critical role in resistance to immunotherapy. In this study, we identified epidermal growth factor-like 6 (Egfl6) as a regulator of myeloid cell functions. Our analyses indicated that Egfl6, via binding with β3 integrins and activation of p38 and SYK signaling, acts as a chemotactic factor for myeloid cell migration and promotes their differentiation toward an immunosuppressive state. In syngeneic mouse models of ovarian cancer (OvCa), tumor expression of Egfl6 increased the intratumoral accumulation of polymorphonuclear (PMN) MDSCs and TAMs and their expression of immunosuppressive factors, including CXCL2, IL-10, and PD-L1. Consistent with this, in an immune 'hot' tumor model, Egfl6 expression eliminated response to anti-PD-L1 therapy, while Egfl6 neutralizing antibody decreased the accumulation of tumor-infiltrating CD206+ TAMs and PMN-MDSCs and restored the efficacy of anti-PD-L1 therapy. Supporting a role in human tumors, in human OvCa tissue samples, areas of high EGFL6 expression colocalized with myeloid cell infiltration. scRNA-Seq analyses revealed a correlation between EGFL6 and immune cell expression of immunosuppressive factors. Our data provide mechanistic insights into the oncoimmunologic functions of EGFL6 in mediating tumor immune suppression and identified EGFL6 as a potential therapeutic target to enhance immunotherapy in patients with OvCa.

The mitochondrial calcium uniporter channel (MCUC) mediates mitochondrial calcium entry, regulating energy metabolism and cell death. Although several MCUC components have been identified, the molecular basis of mitochondrial calcium signaling networks and their remodeling upon changes in uniporter activity have not been assessed. Here, we map the MCUC interactome under resting conditions and upon chronic loss or gain of mitochondrial calcium uptake. We identify 89 high-confidence interactors that link MCUC to several mitochondrial complexes and pathways, half of which are associated with human disease. As a proof-of-concept, we validate the mitochondrial intermembrane space protein EFHD1 as a binding partner of the MCUC subunits MCU, EMRE, and MCUB. We further show a MICU1-dependent inhibitory effect of EFHD1 on calcium uptake. Next, we systematically survey compensatory mechanisms and functional consequences of mitochondrial calcium dyshomeostasis by analyzing the MCU interactome upon EMRE, MCUB, MICU1, or MICU2 knockdown. While silencing EMRE reduces MCU interconnectivity, MCUB loss-of-function leads to a wider interaction network. Our study provides a comprehensive and high-confidence resource to gain insights into players and mechanisms regulating mitochondrial calcium signaling and their relevance in human diseases., (© 2024. The Author(s).)

Peripheral nerve injury (PNI) often leads to significant functional impairment. Here, we investigated the impact of epidermal growth factor-like domain-containing protein 7 (EGFL7) on angiogenesis and nerve regeneration following PNI. Using a sciatic nerve injury model, we assessed nerve function using the sciatic nerve function index. We analyzed the expression levels of EGFL7, forkhead box proteins A1 (FOXA1), nerve growth factor (NGF), brain-derived neurotrophic factors (BDNF), Neurofilament 200 (NF200), myelin protein zero (P0), cell adhesion molecule 1 (CD31), vascular endothelial growth factor (VEGF), and NOTCH-related proteins in tissues and cells. Cell proliferation, migration, and angiogenesis were evaluated through cell counting kit assays, 5-ethynyl-2'deoxyuridine staining, and Transwell assays. We investigated the binding of FOXA1 to the EGFL7 promoter using dual-luciferase assays and chromatin immunoprecipitation. We observed decreased EGFL7 expression and increased FOXA1 expression in PNI, and EGFL7 overexpression alleviated gastrocnemius muscle atrophy, increased muscle weight, and improved motor function. Additionally, EGFL7 overexpression enhanced Schwann cell and endothelial cell proliferation and migration, promoted tube formation, and upregulated NGF, BDNF, NF200, P0, CD31, and VEGF expression. FOXA1 was found to bind to the EGFL7 promoter region, inhibiting EGFL7 expression and activating the NOTCH signaling pathway. Notably, FOXA1 overexpression counteracted the effects of EGFL7 on Schwann cells and endothelial cells. In conclusion, EGFL7 holds promise as a therapeutic molecule for treating sciatic nerve injury., (© 2024 International Federation of Cell Biology.)

As cytoplasmic protein complexes that are pivotal for innate immunity, inflammasomes act primarily through the detection of pathogen- or danger-associated molecular patterns. Nucleotide oligomerisation domain-like receptor family and caspase activation recruitment domain-containing protein 4 (NLRC4) inflammasomes identify and eliminate intracellular pathogens, a process contingent on the ligand-recognition capabilities of neuronal apoptosis inhibitory proteins (NAIPs). Upon detection of specific molecules indicative of intracellular infection, NAIPs discern distinct pathogenic components and subsequently transmit signals to NLRC4, thus initiating their activation and triggering an inflammatory response. However, the mechanisms underlying NLRC4 inflammasome remain unclear. In this study, we elucidated the critical role of ATG16L2 in activating the NLRC4 inflammasome. ATG16L2-deficient macrophages exhibited reduced NLRC4 inflammasome activation, characterised by decreased oligomerisation of apoptosis-associated speck-like protein containing a CARD and attenuated cleavage of Pro-caspase-1, Pro-IL-1β and gasdermin D. Co-immunoprecipitation assays revealed an interaction between ATG16L2 and NAIPs. Furthermore, ATG16L2 enhanced the association between NAIPs and NLRC4 by binding to NAIPs. For ATG16L2-knockout mice infected with Salmonella typhimurium, pathogen clearance and survival rates markedly decreased. Collectively, our findings suggest that ATG16L2 is a significant modulator of the innate immune system, influencing the activity of the NLRC4 inflammasome and the host's defensive response to intracellular pathogens., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Thoracic aortic dissection (TAD) is a severe disease, characterized by numerous apoptotic vascular smooth muscle cells (VSMCs). EDIL3/Del-1 is a secreted protein involved in macrophage efferocytosis in acute inflammation. Here, we aimed to investigate whether EDIL3 promoted the internalization and degradation of apoptotic VSMCs during TAD. The levels of EDIL3 were decreased in the serum and aortic tissue from TAD mice. Global edil3 knockout ( edil3 -/- ) mice and edil3 -/- bone marrow chimeric mice exhibited a considerable exacerbation in β-aminopropionitrile monofumarate (BAPN)-induced TAD, accompanied with increased apoptotic VSMCs accumulating in the damaged aortic tissue. Two types of phagocytes, RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were used for in vitro efferocytosis assay. edil3 -deficient phagocytes exhibited inefficient internalization and degradation of apoptotic VSMCs. Instead, EDIL3 promoted the internalization phase through interacting with phosphatidylserine (PtdSer) on apoptotic VSMCs and binding to the macrophage ITGAV/α v -ITGB3/β 3 integrin. In addition, EDIL3 accelerated the degradation phase through activating LC3-associated phagocytosis (LAP). Mechanically, following the engulfment, EDIL3 enhanced the activity of SMPD1/acid sphingomyelinase in the phagosome through blocking ITGAV-ITGB3 integrin, which facilitates phagosomal reactive oxygen species (ROS) production by NAPDH oxidase CYBB/NOX2. Furthermore, exogenous EDIL3 supplementation alleviated BAPN-induced TAD and promoted apoptotic cell clearance. EDIL3 may be a novel factor for the prevention and treatment of TAD. Abbreviations: BAPN: β-aminopropionitrile monofumarate; BMDM: bone marrow-derived macrophage; C12FDG: 5-dodecanoylaminofluorescein-di-β-D-galactopyranoside; CTRL: control; CYBB/NOX2: cytochrome b-245, beta polypeptide; DCFH-DA: 2',7'-dichlorofluorescin diacetate; EDIL3/Del-1: EGF-like repeats and discoidin I-like domains 3; EdU: 5-ethynyl-2'-deoxyuridine; EVG: elastic van Gieson; H&E: hematoxylin and eosin; IL: interleukin; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAC: N-acetylcysteine; PtdSer: phosphatidylserine; rEDIL3: recombinant EDIL3; ROS: reactive oxygen species; SMPD1: sphingomyelin phosphodiesterase 1; TAD: thoracic aortic dissection; TEM: transmission electron microscopy; VSMC: vascular smooth muscle cell; WT: wild-type.

Background: Hypospadias is a common congenital anomaly characterized by the displacement of the urethral opening to the ventral side of the penis. Surgical correction is often necessary for functional and psychological reasons. The etiology involves genetic and environmental factors, and chordee, a downward curvature of the penis, is a common complication. Proteins such as fibulin-1, elastin, matrix metalloproteinase-1, basic fibroblast growth factor, and α-smooth muscle actin play roles in hypospadias development., Objective: The study's aim is to investigate the differences in messenger RNA (mRNA) expression of fibulin-1, elastin, matrix metalloproteinase-1, basic fibroblast growth factor, and α-smooth muscle actin between the ventral and dorsal tunica dartos in patients with hypospadias and chordee., Methods: This prospective cohort study aims to investigate differences in mRNA expression of the abovementioned proteins between the ventral and dorsal tunica dartos in patients with hypospadias and chordee. Ethics approval has been obtained, and consent from parents will be obtained before data collection. Eligible participants are aged 6-18 months, diagnosed with hypospadias and chordee, and planned for urethroplasty. Tissue samples will be collected from both aspects of the tunica dartos and analyzed using real-time quantitative reverse transcription-polymerase chain reaction. Data analysis will involve statistical tests and normalization of housekeeping genes., Results: This study is at the protocol development stage. A pilot study regarding its feasibility has been ongoing as of August 2023. The study results are expected to be available by the end of 2024., Conclusions: The study of mRNA expressions of various proteins in the tunica dartos of patients with hypospadias and chordee is expected to improve the understanding and expand the knowledge of the pathophysiology of hypospadias and chordee., International Registered Report Identifier (irrid): DERR1-10.2196/52282., (©Joko Pitoyo, Alvarino, Eryati Darwin, Yanwirasti. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 30.10.2024.)

Background: C3H10T1/2 is a mesenchymal cell line capable of differentiating into osteoblasts, adipocytes and chondrocytes. The differentiation of these cells into osteoblasts is modulated by various transcription factors, such as RUNX2. Additionally, several interconnected signaling pathways, including the NOTCH pathway, play a crucial role in modulating their differentiation into mature bone cells. We have investigated the roles of DLK1 and DLK2, two non-canonical inhibitory ligands of NOTCH receptors, in the osteogenic differentiation of C3H10T1/2 cells., Results: Our results corroborate existing evidence that DLK1 acts as an inhibitor of osteogenesis. In contrast, we demonstrate for the first time that DLK2 enhances this differentiation process. Additionally, our data suggest that NOTCH2, 3 and 4 receptors may promote osteogenesis, as indicated by their increased expression during this process, whereas NOTCH1 expression, which decreases during cell differentiation, might inhibit osteogenesis. Moreover, treatment with DAPT, a NOTCH signaling inhibitor, impeded osteogenic differentiation. We have confirmed the increase in ERK1/2 MAPK and p38 MAPK phosphorylation in C3H10T1/2 cells induced to differentiate to osteoblasts. Our new findings reveal increased ERK1/2 MAPK phosphorylation in differentiated C3H10T1/2 cells with a decrease in DLK1 expression or an overexpression of DLK2, which is coincident with the behavior of those transfectants where we have detected an increase in osteogenic differentiation. Additionally, p38 MAPK phosphorylation increases in differentiated C3H10T1/2 cells with reduced DLK1 levels., Conclusions: Our results suggest that DLK1 may inhibit osteogenesis, while DLK2 may promote it, by modulating NOTCH signaling and the phosphorylation of ERK1/2 and p38 MAPK pathways. Given the established inhibitory effect of DLK proteins on NOTCH signaling, these new insights could pave the way for developing future therapeutic strategies aimed at treating bone diseases., (© 2024. The Author(s).)

The mitochondrial Ca2+ uniporter (MCU) plays crucial role in intramitochondrial Ca2+ uptake, allowing Ca2+-dependent activation of oxidative metabolism. In recent decades, the role of MCU pore-forming proteins has been highlighted in cancer. However, the contribution of MCU-associated regulatory proteins mitochondrial calcium uptake 1 and 2 (MICU1 and MICU2) to pathophysiological conditions has been poorly investigated. Here, we describe the role of MICU2 in cell proliferation and invasion using in vitro and in vivo models of human colorectal cancer (CRC). Transcriptomic analysis demonstrated an increase in MICU2 expression and the MICU2/MICU1 ratio in advanced CRC and CRC-derived metastases. We report that expression of MICU2 is necessary for mitochondrial Ca2+ uptake and quality of the mitochondrial network. Our data reveal the interplay between MICU2 and MICU1 in the metabolic flexibility between anaerobic glycolysis and OXPHOS. Overall, our study sheds light on the potential role of the MICUs in diseases associated with metabolic reprogramming., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Robert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Nesfatin-1 is a neuropeptide that regulates the hypothalamic-pituitary-gonadal axis and may play a role in uterus function. It is co-expressed with other peptides, such as phoenixin, which can influence sex hormone secretion. Our previous research has confirmed that phoenixin-14 is involved in the development of cystic endometrial hyperplasia (CEH) and pyometra in dogs. Therefore, based on the similarities and interactions between these neuropeptides, we hypothesized that nesfatin-1 might also regulate the reproductive system in dogs. This study aimed to determine the expression of nesfatin-1 and its interaction with phoenixin-14 in dogs with CEH or pyometra compared to healthy females, and concerning animals' body condition score (BCS 4-5/9 vs. BCS > 5/9)., Results: The analysis of nesfatin-1 in the uterus of bitches consisted of qPCR, western blot and immunofluorescence assays, and ELISAs. The results showed significantly higher nesfatin-1 encoding gene, nucleobindin-2 mRNA (Nucb2) and nesfatin-1 protein expression in overweight females and those suffering from CEH or pyometra compared to healthy animals. The immunoreactivity of nesfatin-1 was elevated in the uteri of bitches with higher BCS > 5/9. Moreover, nesfatin-1 blood concentrations increased in all examined overweight bitches. In the case of phoenixin signals, we found opposite results, regardless of the female body condition score., Conclusion: The etiology of CEH and pyometra are not fully known, although we have expanded the level of knowledge with respect to the possible interaction of nesfatin-1 and phoenixin in female dogs' uteri. They interact oppositely. With increasing female body weight, the expression of nesfatin-1 in the uterus and its peripheral blood concentration increased. However, for female dogs affected by CEH and pyometra, a decreased level of phoenixin-14, irrespective of their body condition score is characteristic. This knowledge could be crucial in the development of biomarkers for these conditions, which may lead to earlier recognition., (© 2024. The Author(s).)

Background: The biological functions of mitochondrial complexes are closely related to the development of atrial fibrillation (AF). Calcium binding and coiled-coil domain 2 (CALCOCO2) is a novel and specific receptor for mitophagy; however, its function in AF remains unknown. Therefore, this study aimed to investigate the role and molecular mechanisms of CALCOCO2 in AF, especially its regulatory mechanism in mitophagy and mitochondrial stress., Methods: Mice and HL-1 cells were treated with AngII to establish in vitro and in vivo AF models. Additionally, we examined the effect of CALCOCO2 or DAP3 Binding Cell Death Enhancer 1 (DELE1) overexpression on mitophagy and mitochondrial stress in AF models. To investigate the role of mitophagy in the regulatory effects of CALCOCO2 in AF, HL-1 cells were treated with chloroquine, a mitophagy inhibitor. Moreover, mitochondrial parameters were examined using specific fluorescent probes, transmission electron microscopy, western blotting, immunohistochemistry, and confocal microscopy., Results: AngII severely impaired the normal morphology and function of mitochondria; inhibited mitophagy; promoted atrial mitochondrial stress, fibrosis, and oxidative stress; and accelerated the progression of atrial remodeling in atrial myocytes. However, CALCOCO2 overexpression reversed/ameliorated these AF-induced changes. Additionally, CALCOCO2 overexpression restored mitochondrial homeostasis in atrial muscle by activating mitophagy and ameliorating mitochondrial stress. Mechanistically, DELE1 overexpression increased mitochondrial reactive oxygen species level and the expression of mitochondrial stress proteins (HRI, eIF2α, and ATF4) even in CALCOCO2-expressing in vitro AF models.., Conclusions: CALCOCO2 may serve as a potential target for AF therapy to prevent or reverse the progression of atrial remodeling by regulating mitophagy and DELE1-mediated mitochondrial stress., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Recurring lung injury, chronic inflammation, aberrant tissue repair and impaired tissue remodelling contribute to the pathogenesis of pulmonary fibrosis (PF). Neutrophil extracellular traps (NETs) are released by activated neutrophils to trap, immobilise and kill invading pathogen and is facilitated by peptidyl arginine deiminase-4 (PAD-4). Dysregulated NETs release and abnormal PAD-4 activation plays a crucial role in activating pro-fibrotic events in PF. Developmental endothelial locus-1 (Del-1), expressed by the endothelial cells of lungs and brain acts as an endogenous inhibitor of inflammation and fibrosis. We have hypothesised that PAD-4 inhibitor exerts anti-inflammatory and anti-fibrotic effects in mice model of PF. We have also hypothesised by PAD-4 regulated the transcription of Del-1 through co-repression and its inhibition potentiates anti-fibrotic effects of Del-1. In our study, the PAD-4 inhibitor chloro-amidine (CLA) demonstrated anti-NETotic and anti-inflammatory effects in vitro in differentiated HL-60 cells. In a bleomycin-induced PF mice model, CLA administration in two doses (3 mg/kg, I.P and 10 mg/kg, I.P) improved lung function, normalized bronchoalveolar lavage fluid parameters, and attenuated fibrotic events, including markers of extracellular matrix and epithelial-mesenchymal transition. Histological analyses confirmed the restoration of lung architecture and collagen deposition with CLA treatment. ELISA, IHC, IF, RT-PCR, and immunoblot analysis supported the anti-NETotic effects of CLA. Furthermore, BLM-induced PF reduced Del-1 and p53 expression, which was normalized by CLA treatment. These findings suggest that inhibition of PAD-4 results in amelioration of PF in animal model and may involve modulation of Del-1 and p53 pathways, warranting further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pyroptosis, an inflammatory programmed cell death, has recently been found to play an important role in spinal cord injury (SCI). C-type lectin domain family 5 member A (CLEC5A), triggering receptor expressed on myeloid cells 1 (TREM1), and NLR-family CARD-containing protein 4 (NLRC4) have been reported to be associated with neuronal pyroptosis, but few studies have clarified their functions and regulatory mechanisms in SCI. In this study, CLEC5A, TREM1, and NLRC4 were highly expressed in lidocaine-induced SCI rat models, and their knockdown alleviated lidocaine-induced SCI. The elevation of pyroptosis-related indicators LDH, ASC, GSDMD-N, IL-18, caspase-1, and IL-1β levels in SCI rats was attenuated after silencing of CLEC5A, TREM1, or NLRC4. Lidocaine-induced decrease in cell viability and the elevation in cell death were partly reversed after CLEC5A, TREM1, or NLRC4 silencing. Lidocaine-mediated effects on the levels of LDH, ASC, GSDMD-N, IL-18, caspase-1, and IL-1β in lidocaine-induced PC12 cells were weakened by downregulating CLEC5A, TREM1, or NLRC4. CLEC5A could interact with TREM1 to mediate NLRC4 expression, thus accelerating neuronal pyroptosis, ultimately leading to SCI exacerbation. In conclusions, CLEC5A interacted with TREM1 to increase NLRC4 expression, thus promoting neuronal pyroptosis in rat SCI models, providing new insights into the role of neuronal pyroptosis in SCI., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Tan et al.)

The role of biomarkers in cancer treatment varies significantly depending on the cancer stage. Thus, in clinical practice, tailoring biomarkers to meet the specific needs and challenges of each cancer stage can increase the precision of treatment. Because they reflect underlying genetic alterations that influence cancer progression, copy number variation (CNV) biomarkers can play crucial prognostic roles. In our previous study, we identified potential survival-related genes for colorectal cancer (CRC) by analyzing CNV and gene expression data using a machine-learning approach. To further investigate the biological function of NRXN1, we assessed the use of RNA sequencing, phosphokinase assays, real-time quantitative PCR, and Western blot analysis. We found that NRXN1 copy number deletion was significantly associated with poor overall survival (OS) and recurrence-free survival (RFS), even in patients who received adjuvant chemotherapy. Compared with its expression in normal tissues, NRXN1 expression was lower in tumors, suggesting its potential role as a tumor suppressor. NRXN1 knockdown enhanced CRC cell viability and invasion, and transcriptome analysis indicated that the increased invasion was caused by GSK3β-mediated epithelial-mesenchymal transition. These findings highlight NRXN1 copy number deletion as a novel biomarker for predicting recurrence and survival in patients with resected colon cancer.

Phospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14Δ-PLN. Complexome profiling (CP) is a proteomics workflow for systematic analysis of high molecular weight (MW) protein complexes and supercomplexes. We hypothesize that R14Δ-PLN may alter a subset of these assemblies, and apply CP workflows to explore these changes in presymptomatic R14Δ/+ mice hearts. Ventricular tissues from presymptomatic 28wk-old WT and R14Δ/+ mice were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) with a linear MW-range exceeding 5 MDa, and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) analysis. Unfortunately, current workflows for the systematic analysis of CP data proved ill-suited for use in cardiac samples. Most rely upon curated protein complex databases to provide ground-truth for analysis; however, these are derived primarily from cancerous or immortalized cell lines and, consequently, cell-type specific complexes (including cardiac-specific machinery potentially affected in R14Δ-PLN hearts) are poorly covered. We thus developed PERCOM: a novel CP data-analysis strategy that does not rely upon these databases and can, furthermore, be implemented on widely available spreadsheet software. Applying PERCOM to our CP dataset resulted in the identification of 296 proteins with disrupted elution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) supercomplex components. Changes to mitochondrial supercomplexes were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. The observed alterations to mitochondrial and ICD supercomplexes were replicated in a second cohort of "juvenile" 9wk-old mice. These early-stage changes to key cardiac machinery may contribute to R14Δ-PLN pathogenesis., Competing Interests: RAdB and HS have received research grants for scientific projects from AstraZeneca, Abbott, Boehringer Ingelheim, Cardior Pharmaceuticals GmbH, Ionis Pharmaceuticals, Novo Nordisk, and Roche. These projects were independent projects and had no direct connection with the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. RAdB furthermore declares speaker fees and advisory board fees for Abbott, AstraZeneca, Bristol Myers Squibb, Cardior Pharmaceuticals GmbH, NovoNordisk, and Roche; RAdB received travel support from Abbot, Cardior Pharmaceuticals GmbH, and NovoNordisk for topics outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Foo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function)., Objective: Our objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles - extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs., Method: Using Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting., Results: DOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs., Discussion: Clonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome., Competing Interests: Part of the methodology used in this work is the subject of a provisional patent application. Patent holders may be entitled to certain compensation through their institutions’ respective intellectual property policies in the event such intellectual property is licensed. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Esparza, Lima, Abuelreich, Ghaeli, Hwang, Oh, Lenz, Gu, Jiang, Kandeel, Thurmond and Jovanovic-Talisman.)

Purpose: Cumulating evidence supports the close association between periodontal diseases, neuroinflammation and neurodegenerative pathologies, except for peri-implantitis (PI). Thus, this study explored the association between experimental PI and neuropathological changes in the rat brain., Materials and Methods: After bilateral first molars extraction, experimental PI was induced at titanium implants placed in the maxillae by lipopolysaccharide injections and ligature placement. Following 28-weeks of disease progression, the maxillae and brains were retrieved from 6 rats. Healthy brains from 3 rats were used as control. Brains were analyzed by immunohistochemistry to detect signs of neuroinflammation (interleukin (IL)-6 and tumor necrosis factor (TNF)-α)), microglial activation (IBA-1) and astrogliosis (GFAP). To explore signs of neurodegeneration, hematoxylin/eosin and Nissl stainings were used. Also, four different antibodies against amyloid beta (Aβ 1-42) were tested., Results: Chronic PI lesions showed peri-implant bone resorption accompanied by large inflammatory infiltrates. IL-6 + and TNF-α + cells were found within the CA1 and Dentate Gyrus regions of the hippocampus of the PI-affected group, while almost no immune-positivity was detected in the control (p < 0.05). Detection of activated GFAP + microglia and IBA-1 + astrocytes surface were significantly higher at the CA areas, and cerebral cortex of the PI-affected group, in comparison with control (p < 0.05). Shrunk neurons with pyknotic nuclei were inconsistently found among the PI-affected group, and these were almost not detected in control. No positive Aβ reactivity was detected in any of the samples., Conclusion: Chronic experimental PI lesions led to an increased detection of IL-6 and TNF-α, GFAP + microgliosis and IBA-1 + astrocytosis, and in some cases, neurodegeneration, in the rat brain., (© 2024. The Author(s).)

Notch signaling, an important signaling pathway in cardiac development, has been shown to mediate the reparative functions of c-kit+ progenitor cells (CPCs). However, it is unclear how each of the four canonical Notch-activating ligands affects intracellular processes in c-kit+ cells when used as an external stimulus. Neonatal c-kit+ CPCs were stimulated using four different chimeric Notch-activating ligands tethered to Dynabeads, and the resulting changes were assessed using TaqMan gene expression arrays, with subsequent analysis by principal component analysis (PCA). Additionally, functional outcomes were measured using an endothelial cell tube formation assay and MSC migration assay to assess the paracrine capacity to stimulate new vessel formation and recruit other reparative cell types to the site of injury. Gene expression data showed that stimulation with Jagged-1 is associated with the greatest pro-angiogenic gene response, including the expression of VEGF and basement membrane proteins, while the other canonical ligands, Jagged-2, Dll-1, and Dll-4, are more associated with regulatory and epigenetic changes. The functional assay showed differential responses to the four ligands in terms of angiogenesis, while none of the ligands produced a robust change in migration. These data demonstrate how the four Notch-activating ligands differentially regulate CPC gene expression and function.

Intermittent hypoxia (IH) has been extensively studied in recent years, demonstrating adverse and beneficial effects on several physiological systems. However, the precise mechanism underlying its cardiac effects on the heart remains unclear. This study aims to explore the effect of treatment on atrial fibrillation under IH conditions, providing data that can potentially be used in the treatment of heart disease. An atrial fibrillation (AF) model was induced by injecting monocrotaline (MCT, 60 mg/kg) into rats. The study included 32 rats divided into four groups: Control, Control + IH, AF, and AF + IH. We evaluated molecular changes associated with AF using ELISA and Western blot and performed electrophysiological experiments to evaluate AF. Arrhythmia-related calcium and fibrosis markers were investigated. Phosphorylation levels of CaMKII, Phospholamban, and RyR2 all increased in the AF group but decreased in the IH-exposed group. Additionally, fibrosis marker expressions such as SMA, MMP2, MMP9, and TGF-β increased in the AF group but were significantly downregulated with IH treatment. Connexin 43 and AQP4 expression were restored in the IH-treated group. These findings suggest that IH may prevent AF by downregulating the expression of calcium-handling proteins and fibrosis-associated proteins in an AF-induced rat model.

During mammalian cardiomyocyte excitation-contraction coupling, Ca2+ influx through voltage-gated Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptor channels. This Ca2+-induced Ca2+ release mechanism controls cardiomyocyte contraction and is exquisitely regulated by SR Ca2+ levels. The histidine-rich calcium-binding protein (HRC) and its aspartic acid-rich paralogue aspolin are high-capacity, low-affinity Ca2+-binding proteins. Aspolin also acts as a trimethylamine N-oxide demethylase. At low intraluminal Ca2+ concentrations, HRC binds to the SR Ca2+-ATPase 2, inhibiting its Ca2+-pumping activity. At high intraluminal Ca2+ levels, HRC interacts with triadin to reduce Ca2+ release through ryanodine receptor channels. This Review analyses the evolution of these Ca2+-regulatory proteins, to gain insights into their roles. It reveals that HRC homologues are present in chordates, annelid worms, molluscs, corals and sea anemones. In contrast, triadin appears to be a chordate innovation. Furthermore, HRC is evolving more rapidly than other cardiac excitation-contraction coupling proteins. This positive selection (or relaxed negative selection) occurs along most of the mammalian HRC protein sequence, with the exception being the C-terminal cysteine-rich region, which is undergoing negative selection. The histidine-rich region of HRC might be involved in pH sensing, as an adaptation to air-breathing, endothermic and terrestrial life. In addition, a cysteine-rich pattern within HRC and aspolin is also found in a wide range of iron-sulfur cluster proteins, suggesting roles in redox reactions and metal binding. The polyaspartic regions of aspolins are likely to underlie their trimethylamine N-oxide demethylase activity, which might be mimicked by the acidic regions of HRCs. These potential roles of HRCs and aspolins await experimental verification., Competing Interests: Competing interests The author declares no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Cytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins., Methods: Given the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle., Results: In affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs., Conclusions: Our study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines., (© 2024. The Author(s).)

Vascular calcification (VC) arises from the accumulation of calcium salts in the intimal or tunica media layer of the aorta, contributing to higher risk of cardiovascular events and mortality. Despite this, the mechanisms driving VC remain incompletely understood. We previously described that nesfatin-1 functioned as a switch for vascular smooth muscle cells (VSMCs) plasticity in hypertension and neointimal hyperplasia. In this study, we sought to investigate the role and mechanism of nesfatin-1 in VC. The expression of nesfatin-1 was measured in calcified VSMCs and aortas, as well as in patients. Loss- and gain-of-function experiments were evaluated the roles of nesfatin-1 in VC pathogenesis. The transcription activation of nesfatin-1 was detected using a mass spectrometry. We found higher levels of nesfatin-1 in both calcified VSMCs and aortas, as well as in patients with coronary calcification. Loss-of-function and gain-of-function experiments revealed that nesfatin-1 was a key regulator of VC by facilitating the osteogenic transformation of VSMCs. Mechanistically, nesfatin-1 promoted the de-ubiquitination and stability of BMP-2 via inhibiting the E3 ligase SYTL4, and the interaction of nesfatin-1 with BMP-2 potentiated BMP-2 signaling and induced phosphorylation of Smad, followed by HDAC4 phosphorylation and nuclear exclusion. The dissociation of HDAC4 from RUNX2 elicited RUNX2 acetylation and subsequent nuclear translocation, leading to the transcription upregulation of OPN, a critical player in VC. From a small library of natural compounds, we identified that Curculigoside and Chebulagic acid reduced VC development via binding to and inhibiting nesfatin-1. Eventually, we designed a mass spectrometry-based DNA-protein interaction screening to identify that STAT3 mediated the transcription activation of nesfatin-1 in the context of VC. Overall, our study demonstrates that nesfatin-1 enhances BMP-2 signaling by inhibiting the E3 ligase SYTL4, thereby stabilizing BMP-2 and facilitating the downstream phosphorylation of SMAD1/5/9 and HDAC4. This signaling cascade leads to RUNX2 activation and the transcriptional upregulation of MSX2, driving VC. These insights position nesfatin-1 as a potential therapeutic target for preventing or treating VC, advancing our understanding of the molecular mechanisms underlying this critical cardiovascular condition., (© 2024. The Author(s).)

Under neuroinflammatory conditions, astrocytes acquire a reactive phenotype that drives acute inflammatory injury as well as chronic neurodegeneration. We hypothesized that astrocytic Delta-like 4 (DLL4) may interact with its receptor NOTCH1 on neighboring astrocytes to regulate astrocyte reactivity via downstream juxtacrine signaling pathways. Here we investigated the role of astrocytic DLL4 on neurovascular unit homeostasis under neuroinflammatory conditions. We probed for downstream effectors of the DLL4-NOTCH1 axis and targeted these for therapy in two models of CNS inflammatory disease. We first demonstrated that astrocytic DLL4 is upregulated during neuroinflammation, both in mice and humans, driving astrocyte reactivity and subsequent blood-brain barrier permeability and inflammatory infiltration. We then showed that the DLL4-mediated NOTCH1 signaling in astrocytes directly drives IL-6 levels, induces STAT3 phosphorylation promoting upregulation of astrocyte reactivity markers, pro-permeability factor secretion and consequent blood-brain barrier destabilization. Finally we revealed that blocking DLL4 with antibodies improves experimental autoimmune encephalomyelitis symptoms in mice, identifying a potential novel therapeutic strategy for CNS autoimmune demyelinating disease. As a general conclusion, this study demonstrates that DLL4-NOTCH1 signaling is not only a key pathway in vascular development and angiogenesis, but also in the control of astrocyte reactivity during neuroinflammation., (© 2024. The Author(s).)

Spread through air spaces (STAS) is a recognized aggressive pattern in lung cancer, serving as a crucial risk factor for postoperative recurrence. However, its phenotype and related spatial structure have remained elusive. To address these limitations, we conducted a comprehensive study based on spatial data, analyzing over 30,000 spots from 14 non-STAS samples and one STAS sample. We observed increased proliferation activities and angiogenesis in STAS, identifying S100P as a potential biomarker for STAS. Furthermore, our investigation into the heterogeneity of STAS tumor cells revealed a subset identified as S100P + TFF1 +, exhibiting a negative impact on patients' survival in public datasets. This subtype exhibited the highest activities in the TGFb and hypoxia, suggesting its potential pro-tumor role within the tumor microenvironment. To assess the role of S100P + TFF1 + tumor cells in therapy response, we included data from two clinical trial cohorts (BPI-7711 for EGFR-TKI therapy and ORIENT-3 for immunotherapy). The presence of S100P + TFF1 + tumor cells correlated with worse responses to both EGFR-TKI therapy and immunotherapy. Notably, TFF1 emerged as a serum marker for predicting EGFR-TKI response. Cell-cell communication analysis revealed that the TGFb signaling pathway was the most activated in S100P + TFF1 + tumor cells, with TGFB2-TGFBR2 identified as the main ligand-receptor pair. This was further validated by multiplex immunofluorescence performed on twenty NSCLC samples. In summary, our study identified S100P as the biomarker for STAS and highlighted the adverse role of S100P + TFF1 + tumor cells in survival outcomes., (© 2024. The Author(s).)

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternally expressed RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3 × polyA termination sequence into the Gtl2 locus. By analyzing RNA-seq data of mouse embryos combined with histological analysis, we found that silencing of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of the fetal liver, resulting in hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of maternally expressed RNAs and activation of paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the aforementioned phenotypes. In conclusion, these findings illuminate a novel mechanism by which the silencing of maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through the activation of apoptosis., (© 2024. The Author(s).)

The 5-lipoxygenase/leukotriene system has been implicated in both physiological and pathological states within the central nervous system. Understanding how this system interacts with the dopaminergic system could provide valuable insights into dopamine-related pathologies. This study focused on examining both motor and non-motor dopamine-related responses in 5-lipoxygenase/leukotriene-deficient mice. We used pharmacological agents such as amphetamine, apomorphine, and reserpine to challenge the dopaminergic system, evaluating their effects on prepulse inhibition reaction (PPI), general motor activity, and oral involuntary movements. Additionally, we analyzed striatal glial marker expression (GFAP and Iba-1) in reserpine-treated mice. The 5-lipoxygenase/leukotriene-deficient mice exhibited increased spontaneous locomotor activity, including both horizontal and vertical exploration, along with stereotyped behavior compared to wild-type mice. This hyperactivity was reduced by acute apomorphine treatment. Although basal PPI responses were unchanged, 5-lipoxygenase/leukotriene-deficient mice displayed a significant reduction in susceptibility to amphetamine-induced PPI disruption. Conversely, these mice were more vulnerable to reserpine-induced involuntary movements. There were no significant differences in the basal expression of striatal GFAP and Iba-1 positive cells between 5-lipoxygenase/leukotriene-deficient and wild-type mice. However, reserpine treatment significantly increased GFAP immunoreactivity in wild-type mice, an effect not observed in 5-lipoxygenase-deficient mice. Additionally, the percentage of activated microglia was significantly higher in reserpine-treated wild-type mice, an effect absents in 5-lipoxygenase/leukotriene-deficient mice. Our findings suggest that 5-lipoxygenase/leukotriene deficiency leads to a distinctive dopaminergic phenotype, indicating that leukotrienes may influence the modulation of dopamine-mediated responses., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The anterior thalamic nuclei are important for cognition, and memory in particular. However, little is known about how the anterior thalamic nuclei are affected in many neurological disorders partly due to difficulties in selective segmentation in in vivo scans, due to their size and location. Post-mortem studies, therefore, remain a valuable source of information about the status of the anterior thalamic nuclei. We used post-mortem tissue to assess the status of the anteroventral thalamic nucleus in Down syndrome using samples from males and females ranging from 22-65 years in age and comparing to tissue from age matched controls. As expected, there was increased beta-amyloid plaque expression in the Down syndrome group. While there was a significant increase in neuronal density in the Down syndrome group, the values showed more variation consistent with a heterogeneous population. The surface area of the anteroventral thalamic nucleus was smaller in the Down syndrome group suggesting the increased neuronal density was due to greater neuronal packing but likely fewer overall neurons. There was a marked reduction in the proportion of neurons immunoreactive for the calcium-binding proteins calbindin, calretinin, and parvalbumin in individuals with Down syndrome. These findings highlight the vulnerability of calcium-binding proteins in the anteroventral nucleus in Down syndrome, which could both be driven by, and exacerbate, Alzheimer-related pathology in this region., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Toxoplasma gondii , the causative parasite of toxoplasmosis, is an apicomplexan parasite that infects warm-blooded mammals. The ability of the calcium-binding proteins (CBPs) to transport large amounts of Ca 2+ appears to be critical for the biological activity of T. gondii . However, the functions of some members of the CBP family have not yet been deciphered. Here, we characterized a putative CBP of T. gondii , TgpCaBP (TGME49&#95;229480), which is composed of four EF-hand motifs with Ca 2+ -binding capability. TgpCaBP was localized in the cytosol and ER of T. gondii , and parasites lacking the TgpCaBP gene exhibited diminished abilities in cell invasion, intracellular growth, egress, and motility. These phenomena were due to the abnormalities in intracellular Ca 2+ efflux and ER Ca 2+ storage, and the reduction in motility was associated with a decrease in the discharge of secretory proteins. Therefore, we propose that TgpCaBP is a Ca 2+ transporter and signaling molecule involved in Ca 2+ regulation and parasitization in the hosts.IMPORTANCECa 2+ signaling is essential in the development of T. gondii . In this study, we identified a calcium-binding protein in T. gondii , named TgpCaBP, which actively regulates intracellular Ca 2+ levels in the parasite. Deletion of the gene coding for TgpCaBP caused serious deficits in the parasite's ability to maintain a stable intracellular calcium environment, which also impaired the secretory protein discharged from the parasite, and its capacity of gliding motility, cell invasion, intracellular growth, and egress from host cells. In summary, we have identified a novel calcium-binding protein, TgpCaBP, in the zoonotic parasite T. gondii , which is a potential therapeutic target for toxoplasmosis., Competing Interests: The authors declare no conflict of interest.

Cardiovascular complications are the major cause of diabetes mellitus-related morbidity and mortality. Increased renin-angiotensin-aldosterone system activity and decreased β-adrenergic receptor (β-AR) responsiveness contribute to diabetic cardiac dysfunction. We evaluated the effect of sacubitril/valsartan (neprilysin inhibitor plus angiotensin receptor antagonist combination) and valsartan treatments on the diabetic cardiac function through β-AR responsiveness and on protein expression of diastolic components. Six-week-old male Sprague Dawley rats were divided into control, diabetic, sacubitril/valsartan (68 mg/kg)-, and valsartan-treated (31 mg/kg) diabetic groups. Diabetes was induced by a high-fat diet plus low-dose streptozotocin (30 mg/kg, intraperitoneal). After 10 weeks of diabetes, rats were treated for 4 weeks. Systolic/diastolic function was assessed by in vivo echocardiography and pressure-volume loop analysis. β-AR-mediated responsiveness was assessed by in vitro papillary muscle and Langendorff heart experiments. Protein expression of sarcoplasmic reticulum calcium ATPase2a, phospholamban, and phosphorylated phospholamban was determined by Western blot. Sacubitril/valsartan improved ejection fraction and fractional shortening to a similar extent as valsartan alone. None of the treatments affected in vivo diastolic parameters or the expression of related proteins. β 1 -/β 2 -AR-mediated responsiveness was partially restored in treated animals. β 3 -AR-mediated cardiac relaxation (an indicator of diastolic function) responses were comparable among groups. The beneficial effect of sacubitril/valsartan on systolic function may be attributed to improved β 1 -/β 2 -AR responsiveness.

Objective To explore the mechanism of butylphthalide (NBP) in regulating microglia activation and inflammatory cytokine expression in the hippocampus of the mouse model of delayed encephalopathy after carbon monoxide poisoning (DEACMP). Methods Wild-type C57 adult mice with normal cognitive function were selected,and DEACMP was modeled by static inhalation of carbon monoxide.The mice were randomized into three groups:DEACMP,control,and NBP.The NBP group was administrated with NBP suspension at 6 mg/kg by gavage for 21 days,and the DEACMP and control groups were administrated with the same amount of vegetable oil by gavage.The hippocampal injury was observed by HE staining.The protein level of ionized calcium-binding adapter molecule 1 (IBA1) was determined by Western blotting,and the levels of downstream inflammatory cytokines were measured by ELISA. Results Compared with the control group,the DEACMP and NBP groups showed prolonged escape latency ( P =0.001, P =0.029),reduced nerve cells ( P =0.001, P =0.035),up-regulated expression of IBA1 ( P =0.001, P =0.042),increased mean fluorescence intensity of IBA1 ( P =0.001, P =0.021),and elevated levels of tumor necrosis factor-α (TNF-α) ( P =0.002, P =0.024),interleukin (IL)-6 ( P =0.001, P =0.015),and IL-1β ( P =0.001, P =0.023).Compared with the DEACMP group,the NBP group showed shortened escape latency ( P =0.025),increased nerve cells ( P =0.039),down-regulated expression of IBA1 ( P =0.035),decreased average fluorescence intensity of IBA1 ( P =0.031),and lowered levels of TNF-α ( P =0.028),IL-6 ( P =0.037),and IL-1β ( P =0.034). Conclusion NBP can inhibit the activation of microglia and reduce the expression of inflammatory factors,thereby alleviating cognitive dysfunction and brain tissue damage caused by DEACMP.

Partial hepatectomy (PH) is commonly used to treat patients with hepatocellular carcinoma. The recovery of patients from PH depends on the initiation of liver regeneration, a process that mainly relies on liver cell proliferation. As sex affects the human liver regeneration progress, we investigated sex disparity in PH-induced liver regeneration in adult zebrafish. We found that, after PH, males began liver regeneration earlier than females in terms of liver cell proliferation and liver mass recovery, and this was associated with earlier activation of Yap1 signaling in male than female livers. We also found that androgen receptors regulated the sex-biased liver regeneration in a Yap1-dependent manner and that activated estrogen receptors are responsible for the later onset of female hepatocyte proliferation. Furthermore, we identified that S100A1, a calcium-binding protein, regulates the sex disparity in liver regeneration, as heterozygous S100A1 knockout inhibited Yap1 activity in male livers and delayed hepatocyte proliferation in males following PH. Thus, multiple pathways and/or their interplays contribute to the sex disparity in liver regeneration, suggesting that sex-biased therapeutic strategies are required for patients who have received PH-based therapies., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Alpha toxin has become the subject of research in recent years. The objective of this article was to review and summarize recent research on the molecular structure and biological function of the alpha toxin of Clostridium perfringens . This includes the work of our research team, as well as that of other researchers. Clostridium perfringens is an anaerobic, spore-forming, Gram-positive bacillus. It can cause various intestinal diseases, such as gas gangrene, food poisoning, non-foodborne diarrhea, and enteritis. Clostridium perfringens can be classified into 5 toxinotypes A, B, C, D, and E, based on the production of major toxins. Each type of C. perfringens produces alpha toxin, which is one of the most important lethal and dermonecrotic toxins and is considered a primary virulence factor. Alpha toxin is a multifunctional metalloenzyme with phospholipase C and sphingomyelinase activities that simultaneously hydrolyze phosphatidylcholine and sphingomyelin. It can therefore destroy the integrity of cell membranes and eventually cause cell lysis. The clinical effects of alpha toxins are characterized by cytotoxicity, hemolytic activity, lethality, skin necrosis, platelet aggregation, and increased vascular permeability. Future research will concentrate on the pathogenesis of a lpha toxin exposure, clarifying the interaction between alpha toxin and the cell membrane and investigating the mechanism of activating platelet function. This research will have substantial theoretical and practical value in controlling disease progression, identifying targeted therapeutic sites, and reducing the toxic effects of vaccines., Competing Interests: Conflict of interest: The authors declare that there is no conflict of interest associated with the publication of this article., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background/aim: The function of the S-100 protein family member hornerin (HRNR) in gastric cancer (GC) tissues is largely unknown. We researched the clinical significance of HRNR expression in GC tissues of patients with pathological (p)Stage II/III GC after curative resection., Patients and Methods: We included patients with pStage II/III GC who underwent curative gastrectomy (n=253). Expression levels of HRNR in GC tissue and in the adjacent normal mucosa were determined using quantitative real-time polymerase chain reaction. Clinicopathological features and overall survival (OS) were compared in patients with different HRNR expression levels in GC tissue., Results: HRNR expression levels were significantly higher in GC tissues than in the adjacent normal mucosa. HRNR expression level in GC tissue showed sex differences. The 5-year OS rate in the high-HRNR expression group was significantly worse than that in the low-expression group (5-year survival 53.6% vs. 74.9%; p=0.004). Furthermore, on multivariate analysis, high-HRNR expression was an independent predictor of poor OS (hazard ratio=1.534; 95% confidence interval=1.130-2.618; p=0.011)., Conclusion: In patients with pStage II/III GC after curative gastrectomy, HRNR expression in GC tissue may be a useful prognostic marker., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)