Biochemistry and Biophysics Reports

Objective Based on the bioinformatics method of multi-database integration, this study systematically analyzes the expression characteristics, clinical pathological correlation, prognostic value, potential molecular mechanisms, and immune infiltration patterns of hepatocyte growth factor receptor (MET) in gastric cancer. Methods The UALCAN and GEPIA databases were employed to examine the differential expression of MET between gastric cancer and normal gastric mucosal tissues, as well as its associations with clinicopathological features. Kaplan-Meier Plotter was utilized to evaluate the impact of MET expression on overall survival (OS) and progression-free survival (PFS). Protein-protein interaction (PPI) network was constructed via LinkedOmics, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-expressed genes. Four algorithms, TIMER, CIBERSORT, EPIC, and MCPcounter, were used to cross evaluate the correlation between MET expression and immune cell infiltration; Further validate the cell type specific expression of MET using the gastric cancer single-cell sequencing queues (GSE134520, GSE167297) built into the TISCH database. Results MET expression was significantly elevated in gastric cancer tissues compared with normal gastric mucosa (P < .05), and its expression level was significantly correlated with tumor grade and TNM stage. Patients with high MET expression exhibited significantly poorer OS and PFS than those with low MET expression (P < .05). The PPI network revealed that MET could interact with 20 key proteins, including EGFR, ERBB2, HGF, STAT3, and GRB2 etc. GO enrichment analysis suggests that differentially expressed genes are significantly enriched in functions such as the ERBB signaling pathway, cadherin binding, and DNA repair complexes; KEGG enrichment analysis showed that MET related genes were significantly enriched in pathways such as homologous recombination, nuclear cytoplasmic transport, mismatch repair, and oxidative phosphorylation. Immune infiltration analysis showed that the negative association between MET and B cells infiltration has cross algorithm robustness, while the association with neutrophils, CD8+ T cells, CD4+ T cells, and macrophages exhibits algorithmic heterogeneity or insignificance; There is no significant correlation between MET and common immune checkpoint molecules such as PD-1, PD-L1, CTLA4, etc. Single cell validation further confirmed that MET is mainly enriched in malignant epithelial cells and endothelial cells, and is almost not expressed in immune cells. Conclusions Multidimensional bioinformatic analyses demonstrate that elevated MET expression serves as an independent risk factor for unfavorable prognosis in gastric cancer. MET may mediate dual drug resistance in gastric cancer via crosstalk with multiple signaling molecules (including EGFR, ERBB2, HGF, STAT3 and GRB2) and dysregulation of the homologous recombination repair pathway. Results from multiple-algorithm immune infiltration analysis, single-cell dataset analysis and immune checkpoint correlation analysis indicate that MET exerts only modest direct regulatory effects on the gastric cancer immune microenvironment. This exploratory study offers systematic bioinformatic evidence supporting MET as a candidate prognostic biomarker and potential therapeutic target for gastric cancer. Further functional experiments and prospective cohort studies are required to validate its molecular mechanisms and clinical utility.

Breast cancer is a heterogeneous disease comprising molecular subtypes with distinct therapeutic vulnerabilities. Among emerging therapeutic strategies, copper-based nanoparticles have shown anticancer activity. However, whether these responses differ across breast cancer subtypes and which molecular programs underlie copper sensitivity remain poorly understood. We investigated subtype-specific responses to Polyvinylpyrrolidone-assisted copper nanoparticles (CuNP-PVP) in breast cancer cell lines and the molecular programs underlying copper adaptation. CuNP-PVP were synthesized and evaluated in luminal (MCF-7), HER2-positive (SKBR3), and triple-negative (MDA-MB-231) breast cancer cell lines, with transcriptomic analyses integrated with CCLE and TCGA-BRCA datasets. UV-Vis spectroscopy revealed a plasmon resonance band at 591 nm, while TEM showed spherical nanoparticles with an average diameter of 76.1 +/- 21.0 nm. DLS indicated a larger hydrodynamic diameter, consistent with PVP coating; {zeta}-potential measurements showed a surface charge of -13 mV, and XPS confirmed mixed reduced and Cu(I)/Cu(II) oxidation states. Exposure to 100 g/mL CuNP-PVP induced subtype-dependent effects on cell viability, with MCF-7 showing greater sensitivity, whereas MDA-MB-231 and SKBR3 were less affected. Baseline transcriptomic analyses revealed enrichment of oxidative stress, metabolic adaptation, and lysosomal programs in less sensitive cells, whereas MCF-7 showed higher expression of lipoylation-related genes. These features supported the development of a Potential Biological Copper-Response Index (PBCRI), which captured subtype-associated transcriptional patterns across independent cell lines and patient tumors, with luminal models showing lower scores and basal/TNBC models showing higher scores. Together, these findings identify candidate transcriptional programs associated with subtype-specific responses to CuNP and provide a framework for investigating potential molecular determinants of copper sensitivity.

Ovarian cancer stands out as one of the most lethal gynecological cancers among women in the United States. While patients with ovarian cancer often show positive responses to treatment, it is common for the cancer to reappear and develop resistance to treatment, so it is important to explore and create novel combinations to increase patient survival rates. ATRi (Ataxia telangiectasia and Rad3-related inhibitors) and ONC201 monotherapies have shown efficacy in ovarian cancer in preclinical models. We hypothesized that combining ATRi with ONC201 would enhance this efficacy. ONC201 induces apoptosis through the TRAIL-pathway following activation of the integrated stress response and inactivation of Akt which is upregulated in ovarian cancer. Six human ovarian cancer cell lines were treated alone and with the novel drug combination of ceralasertib (ATRi) and ONC201. We found an IC50 range of 0.88-20.16 microM for ONC201 and IC50 range of 0.55-54.75 microM for ceralasertib. The combination of ONC201 plus ceralasertib resulted in PARP cleavage consistent with apoptosis along with reduction in Bcl-XL, Xiap and p-Akt expression after 72 hours of treatment. Cytokine profiling showed a decrease in markers of inflammation and tumor progression. Our data provide evidence of synergy from the combination of ATRi ceralasertib and ONC201 against Ovarian cancer cells that merits further investigation including clinical translation.

Heavy metal contamination of aquatic environments poses persistent risks to ecosystems and human health. Aquatic macrophytes such as Pistia stratiotes are increasingly used for phytoremediation due to their capacity to accumulate contaminants without displaying symptoms of abiotic stress. However, the molecular mechanisms underlying their metal tolerance remain poorly understood. Metallothioneins (MTs) are cysteine-rich proteins involved in metal binding and detoxification in plants. In this study, genome-wide analysis was performed to identify and characterise candidate MT genes in P. stratiotes and to assess their transcriptional response to copper exposure. Six novel MT genes (PsMT2f, PsMT3a-d, and PsMT4) were identified and classified into MT2, MT3, and MT4 subfamilies based on conserved cysteine motifs and domain architecture. Quantitative real-time PCR showed differential and tissue-specific expression of MT genes in roots and leaves following exposure to copper for up to 4 h. These findings provide the first comprehensive characterisation of the MT gene family in P. stratiotes and demonstrate their potential role in copper tolerance, supporting the application of this species in the phytoremediation of metal-contaminated aquatic systems.

Vertical transmission of SARS-CoV-2 is controversial. Owing to the low expression of ACE2 along with TMPRSS2 in placenta, role of secondary receptors becomes significant. In our study we predicted twelve interactors of spike protein expressed in placenta through interactome analysis followed by docking and molecular dynamic simulation. Three interactors of SARS-CoV-2 spike protein were found to be CD147, EGFR and LDLR. Expression at transcriptomic and translational level by real-time PCR and immunohistochemistry respectively was observed in placenta samples obtained from women with COVID exposure risk and compared to control. Elevated protein levels of CD147 (OR=14.667), ACE2 (OR=8.25) and LDLR (OR=1.9) was associated with higher exposure risk. However, the variation in mRNA expression was not statistically significant. Computational study showed that CD147/basigin, EGFR and LDLR stably interact with spike protein outside its primary receptor binding domain. In-vitro binding assay confirmed the physical interaction between spike protein and the placental receptors. In-silico site-directed mutagenesis further identified key potential binding hotspots of spike protein- Tyr28 and Trp64 for CD147, Phe643, Val642, Thr632 for LDLR and Leu8, Phe59, Phe32, Tyr28, Trp64, Trwp152, His245, and Val213 for EGFR. Finally, concluded that CD147, EGFR and LDLR might act as secondary non-canonical cofactors for SARS-CoV-2 spike protein in placenta, thereby increasing its susceptibility and tissue tropism. The study overall gives a better insight into potential placental suscpetibility providing targeted therapeutic opportunities.

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

Antifreeze proteins and peptides adhere to ice crystals and inhibit their growth, resulting in freezing point depression. AFP6 is an abundant antifreeze peptide in the blood serum of winter flounder (Pseudopleuronectes americanus). This 37-residue peptide forms an amphiphilic -helix with a hydrophobic ice-binding surface. In this study, prediction of the peptide structure suggested the formation of an antiparallel dimer burying the hydrophobic surfaces of both subunits. The oligomerisation of AFP6 was therefore examined in aqueous solutions using size-exclusion chromatography and chemical crosslinking along with ice-binding evaluation. Crosslinking of non-amidated AFP6, followed by denaturing electrophoresis, revealed bands consistent with monomers and dimers, whereas results for the amidated form and a recombinant mutant suggested additional larger forms. Bands consistent with the presence of monomers and dimers were also predominant following non-amidated AFP6 crosslinking under a variety of solution conditions, with smaller proportions of peptide assembled up to tetrameric sizes; however, the larger oligomers appeared to be absent from AFP6 treated with sodium bicarbonate. Isolated oligomers of non-amidated AFP6 produced ice crystal morphologies and thermal hysteresis comparable to those for the unfractionated peptide suggesting similar interaction with ice. Oligomerisation of AFP6 may allow greater solubility of this amphiphilic -helical peptide in aqueous solutions.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

Purpose: Uveal melanoma (UM) is a rare but aggressive intraocular malignancy originating in the uvea (choroid, iris, and ciliary body). Despite primary treatment, 50% of UM patients develop metastases to the liver, lungs, bones, and skin. Topotecan, a topoisomerase I inhibitor, and SP141, an MDM2 inhibitor, have been studied in cancers, including retinoblastoma. This study explores a combined approach with these drugs in the 92-1 UM cell line. Methods: Primary choroidal melanoma-derived 92-1 cells were treated with topotecan and SP141 individually to determine IC50 values. Cell viability and proliferation were assessed using the MTT assay and the xCELLigence real-time monitoring system. The synergistic effects were analyzed quantitatively using the Loewe Additivity and Highest Single-Agent (HSA) models. Flow cytometry was employed to analyze cell cycle distribution, while cell death and apoptosis were monitored via high-throughput, real-time quantitative analysis using the IncuCyte live-cell kinetic imaging system. Results: Both Topotecan and SP141 induced significant cell death in 92-1 cells. xCELLigence showed dose- and time-dependent inhibition of proliferation. Combining sub-IC50 concentrations of topotecan and SP141 exhibited significant synergistic effects, markedly reducing growth. Cell cycle analysis revealed that topotecan causes a dose-dependent biphasic arrest in G2 and S phases, while SP141 causes G2/M arrest, leading to cell death. Cytotoxicity assays corroborated increased cell death following combination therapy, and the Caspase 3/7 assay indicated that apoptosis is the mechanism underlying cell death. Conclusions: The multi-targeted approach combining topotecan and SP141 drugs demonstrated synergistic therapeutic effects in 92-1 cells, suggesting a potential treatment strategy for managing UM.

Objectives Low grade serous ovarian cancer (LGSOC) is characterized by frequent TP53 wild type status and limited responsiveness to conventional chemotherapy. This study investigated the cytotoxic effects of oxaliplatin combined with 5-fluorouracil on patient derived LGSOC cells. Results Clinically relevant concentrations of oxaliplatin and 5-fluorouracil had minimal effects on LGSOC cell viability but markedly reduced cellular proliferation and clonogenic recovery. Combined treatment induced pronounced accumulation of cells in the G0/G1 phase of the cell cycle and increased expression of p53 and p21, accompanied by reduced pRB phosphorylation, consistent with activation of the p53-p21-pRb pathway. Oxaliplatin plus 5-fluorouracil also increased senescence-associated beta-galactosidase activity, cellular enlargement and flattening, and reactive oxygen species production, supporting a senescence-like phenotype. However, after drug withdrawal, treated cells progressively regained proliferative capacity, indicating that the senescence phenotype was transient rather than fully irreversible. Together, these findings show that oxaliplatin plus 5-fluorouracil primarily arrest rather than kill LGSOC cells and induces a transient senescence-like phenotype and G0/G1-phase arrest by activating the p53-p21-pRb pathway.

Purpose: There is an unmet need in the treatment of triple-negative breast cancer (TNBC), as recurrence rates remain high; thus, novel treatment strategies are warranted to improve outcomes for patients. Smac mimetics (SMs) are small molecules that inhibit the inhibitors of apoptosis. In this study, we investigated the properties of the SM Xevinapant (AT406) as a radiosensitizer in TNBC. Methods: TNBC cell lines were treated with Xevinapant (AT406) in the presence or absence of ionizing RT and cell viability and apoptosis measured in vitro. Clonogenic assays were performed to confirm radiosensitization. We also assessed xevinapant radiosensitization in the presence of the Tumor Necrosis Factor (TNF) cytokine, which has been shown to enhance SM-induced apoptosis. Radiosensitization mechanisms were further validated by western blotting, flow cytometry and cell cycle analysis. Results: We show that Xevinapant increased cell death in a panel of irradiated TNBC cell lines. In clonogenic assays, Xevinapant sensitized MDA-MB-231 cells to RT-induced cell death, and this was significantly enhanced in the presence of TNF, with a dose-enhancing factor (DEF) of 1.57. We also show that Xevinapant radiosensitization is mediated by TNF-dependent apoptosis. Conclusions: Xevinapant is a radiosensitizer that enhances RT-induced cell death in TNBC cells, and TNF is central to this process through activation of apoptosis. These results are promising for combining RT with Xevinapant to improve local control in TNBC.

Background. Cancer cachexia is a multifactorial syndrome characterized by substantial metabolic disturbances including systemic inflammation, and muscle atrophy, which may affect the regulation of iron metabolism in patients. This study aimed to characterize this regulation in two murine models of colorectal and pancreatic cancer, both associated with a high prevalence of cachexia. Methods. Histological, biochemical and molecular analyses were performed in serum, skeletal muscle, liver, spleen and tumor from UN-KC-6141 pancreatic tumor bearing mice and from ApcMin/+ mice (colorectal cancer model), both models exhibiting variable cachexia severity. Results. Both models induce cachexia, but diverge markedly in their regulation of iron metabolism. UNKC mice display reduced plasma iron availability (-9.6% transferrin saturation, p<0.001) without concomitant anemia, while hepatic hepcidin synthesis is upregulated (+100.6%, p<0.001) by inflammation, promoting ferroportin (FPN) downregulation (-42.4%, p<0.001) and iron sequestration in the spleen (+134% total iron content, p<0.001). In the atrophied gastrocnemius, iron concentration is preserved through increased ferritin (+86.6%, p=0.010) and hemoproteins (i.e., myoglobin, CYSC, COXIV) expression. Conversely, ApcMin/+ mice exhibit anemia together with hepatic and splenic iron depletion (-48.4%, p<0.001 and -84.9%, p<0.001, respectively). Hepcidin synthesis is downregulated (-99.9%, p<0.001) despite persistent inflammation, indicating that hypoxia and/or iron-deficiency signals probably predominate over the inflammatory pathway. FPN is markedly decreased (-94.2%, p<0.001) in the iron-depleted spleen despite low hepcidin. In the atrophied quadriceps of ApcMin/+ mice, iron deficiency is associated with decreased FPN and ferritin (-87.9%, p<0.001, and -38.4%, p=0.016, respectively), and increased TfR1 expression (+305%, p=0.107). Finally, tumor iron import may play a significant role in cachexia progression, as suggested by the association between tumor weight, tumor TfR1 protein levels and body weight loss in UNKC mice. Conclusion. These findings reveal cancer type-specific signatures of iron regulation in cachexia, which appear to be more driven by cancer type specific features than by cachexia severity itself, with skeletal muscle iron metabolism impaired mainly when cachexia is associated with anemia. This suggests that the clinical management of iron metabolism in cachectic patients should be guided by the underlying cancer type.

Background: Epidemiological studies have highlighted an inverse association between cancer and Alzheimers disease (AD), but the mechanisms underlying this association are not currently understood. Amyloid precursor protein (APP) and its cleavage may represent one potential mechanism. Its importance has been heavily researched due to it giving rise to the production of amyloid-beta, a pathological hallmark of AD. APP, however, has not been extensively investigated in cancer. Methods: FFPE prostate tumour and adjacent benign tissue immunohistochemically stained for APP, androgen receptor and the insulin-like growth factor-I receptor (IGF1R). Normal prostate PNT2, androgen sensitive LNCaP and androgen insensitive PC3 in vitro were used to assess the effects of androgen, IGF-I and their inhibitors on APP and its cleavage; changes in protein abundance were measured by western blot; interactions between proteins using co-immunoprecipitation and effects on cell proliferation, apoptosis and cell signalling of APP silenced cells using a BrdU proliferation assay, Muse Cell Analyser and by TMT proteomic analysis respectively. Results: Data showed that APP was upregulated in prostate tumour tissue compared to adjacent benign. Furthermore, in cancer cell models, in contrast to normal prostate epithelial cells, it was observed that APP was critical for prostate cancer cell survival by interacting with androgen and IGF-I signalling pathways. Conclusions: Drugs that target APP have already been developed for AD and our data suggests they offer an exciting opportunity for repurposing in the treatment of prostate cancer.

Introduction: With global warming, heatwaves are increasing in severity, affecting species worldwide. Recent studies show associations between heat exposure in pregnancy and adverse birth outcomes such as preterm birth, low birthweight and stillbirth across multiple species. This suggests potentially conserved mechanisms such as maternal inflammation, dehydration and changes in placental physiology in response to heat exposure. Moreover, the gestational timing of antenatal heat likely impacts the type and severity of outcome, yet this is not fully understood. Methods: We conducted a scoping review of studies on heat exposure and pregnancy outcomes in humans and other animals, to identify common mechanisms and temporal dynamics of effects. Temperature-, trimester- and species- specific patterns were reviewed across 197 studies and ten species (human, cow, sheep, pig, goat, rat, mouse, guinea pig, lizard and rabbit). Results: Qualitative trends indicate that high temperatures are associated with adverse birth outcomes. In particular, third trimester exposure increases risk of preterm birth and low birthweight. Many studies emphasise placental involvement in adverse outcomes and point to potential aberrations in placental physiology or cortisol signaling pathways. Conclusions: Overall, literature presents that heat exposure increases risk of adverse birth outcomes across studies, and taxa. Understanding the relationship between heat exposure and adverse birth outcomes, as well as the mechanisms and timing, is vital for informing public health policies and creating mitigating strategies for expecting mothers.

Ferroptosis has attracted significant attention for its potential to treat drug resistant tumors, along with its role in neurodegeneration and ischemia. Several ferroptosis targets were identified in the past decade, yet it is also highly affected by cellular metabolism and epithelial or mesenchymal phenotypes which limits the utility of these targets across diverse cell and tissue types. To address this, we utilized chemo-resistant cancer cell lines with different metastatic potential and tissue origins and discovered a common role of cytoplasmic ion changes during ferroptosis. We observed significant changes in intracellular potassium, sodium and chloride levels upon induction of ferroptosis. Therefore, we screened several ion channel inhibitors and activators (i.e. modulators) and identified Kv7.2 and CFTR modulators influence ferroptosis execution despite the fact that these cell lines do not express these proteins. We demonstrated that clinically approved modulators of CFTR and Kv7.2 can inhibit ferroptosis which indicates that these inhibitors could be repurposed as treatments in ferroptosis-associated diseases such as neurodegeneration and kidney diseases independent of ion channel expression. These modulators also influenced potassium and chloride levels in different contexts indicating off-target effects may also lead to changes in ion levels and may serve as a ferroptosis target. Further, we demonstrated that sustained imbalance of sodium, potassium and chloride ions with ferroptosis inducer salinomycin and digoxin, a Na+/K+ ATPase inhibitor, enhanced ferroptosis. The combination of salinomycin and digoxin successfully enhanced ferroptotic cell death across both cell lines with different metabolic signatures in vitro, offering a novel ferroptosis target. This combination significantly reduced tumor growth in vivo compared with the single agent alone, offering a new therapeutic modality against drug resistant tumors.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Ultraviolet-C (UV-C) radiation is a potent environmental stressor that induces DNA damage, accumulation of reactive oxygen species (ROS), and cellular dysfunction. This also initiates coordinated molecular responses to regulate cellular adaptation and recovery. Small extracellular vesicles (sEVs) have emerged as key mediators of intercellular communication through the selective incorporation of microRNAs (miRNAs), proteins, and lipids. However, the temporal changes of sEV-associated cargo during the progression from UV-C-induced damage to recovery remain uncharacterized. We hypothesised that UV-C exposure induces stage-specific changes in sEV cargo (miRNA and proteins) that promote the transition of cells from damage to the response and recovery phases. To examine this, a time-resolved UV-C exposure model was established using Caenorhabditis elegans (C. elegans). In this paper, we studied 2.5 hours of UV-C exposure, including cellular damage, intracellular ROS levels, and DNA integrity, to define the damage and repair state. Based on these temporal variations, samples were collected: i) immediately after exposure, ii) 6 hours post-exposure, and iii) 16 hours post-exposure were designated as damage, response, and recovery conditions, respectively. sEVs isolated from whole-worm preparations were characterised using biophysical and molecular approaches, followed by miRNA sequencing and proteomic analyses. Integrative analysis further identified miRNA-protein associations and associated them with pathways involved in stress adaptation, metabolism, signal transduction, and cellular homeostasis. Collectively, these findings provide a temporal molecular framework of sEV cargo associated with UV-C-induced damage, response, and recovery in C. elegans. Overall, this study identified candidate molecular signatures and miRNA-protein associations that underlie the temporally selective packaging of sEV-associated cargo following UV-C-induced damage, providing a basis for future tissue-specific and mechanistic investigations.

Introduction: The femoropopliteal artery undergoes substantial axial compression, bending, torsion, and pinching during limb flexion. When an implanted stent does not accommodate these deformations, repetitive biomechanical mismatch may alter the vascular response to implantation and contribute to restenosis. We tested whether designing a peripheral stent to better accommodate physiologic limb-flexion deformation reduces the subsequent inflammatory and neointimal response. Methods: A nitinol stent was developed using multi-objective computational optimization to maintain arterial apposition while reducing wall stress and luminal pinching during physiologic deformation. Sixteen Yucatan minipigs were studied in independent 3- and 6-month cohorts (n=8 each). Each animal received the optimized stent in one limb and a commercial stent with representative biomechanical behavior contralaterally. CTA quantified in vivo limb-flexion deformation, while CT and histology assessed stenosis, neointimal accumulation, inflammatory-cell burden, SMA-positive tissue, and extracellular-matrix composition. Results: The optimized stent accommodated greater axial shortening than the commercial comparator at both 3 months (11.2{+/-}2.7% vs. 6.6{+/-}2.1%, p=0.003) and 6 months (13.7{+/-}4.4% vs. 6.8{+/-}2.3%, p=0.004). Intimal hyperplasia volume was lower with the optimized stent at 3 months (227.5{+/-}85.2 vs. 350.0{+/-}130.5 mm3, p=0.002) and 6 months (183.6{+/-}46.2 vs. 261.2{+/-}60.6 mm3, p=0.001), with significantly lower maximum, mean, and median stenosis at both timepoints. At 3 months, macrophage and total inflammatory-cell counts were 51% and 45% lower, respectively, with the optimized stent. Histologic tissue area was 28% lower at 3 months and 12% lower at 6 months. SMA-positive tissue fraction was higher with the optimized stent at both timepoints, whereas elastin, glycosaminoglycan, and collagen fractions were similar between devices. Discussion: Improved accommodation of limb-flexion deformation was associated with a smaller inflammatory and neointimal response to stent implantation through 6 months, while overall neointimal matrix composition remained largely unchanged. These findings provide in vivo evidence that biomechanical compatibility with the dynamic lower-limb environment can influence vascular healing and should be considered a mechanistic design criterion for peripheral artery stents.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Background: Transcardial perfusion fixation is widely used to preserve rodent brains for histological and ultrastructural analysis, but protocols can vary in the use of pre-fixation washout, fixative formulation, and osmotic additives. Relatively few studies have directly compared how the chemical composition of the perfusate affects tissue preservation. Methods: Male Sprague-Dawley rats underwent transcardial perfusion using a series of aldehyde-based fixative formulations that varied in fixative composition, use of a phosphate buffered saline washout, and the addition of mannitol or polyethylene glycol 35 kDa (PEG35). Perfusion outcomes were assessed using gross brain morphology, semi-quantitative grading of vascular blood clearance, quantitative detection of residual erythrocytes in whole-slide histological images, and light microscopic measures of cellular visualization and morphology. Perfusate osmolality was measured, and selected specimens were examined by electron microscopy. Results: Substantial vascular blood clearance was frequently achieved with fixative-only perfusion, indicating that a pre-fixation washout was not required to achieve high levels of blood clearance under the conditions tested. Routine light microscopy-based measures of cellular visibility and morphology were broadly similar across treatment groups. Adding mannitol and PEG35 to the fixative solution both led to concentration-dependent gross tissue shrinkage, particularly with PEG35, without correspondingly large or consistent changes on light microscopy. A substantial degree of case-to-case variation in perfusion quality was also observed among animals undergoing nominally similar procedures. Conclusions: Substantial variation in perfusate composition produced relatively modest differences in the light microscopy outcomes examined, despite pronounced effects of osmotic additives on gross brain morphology. There were some cases where perfusion with fixative alone led to near-complete blood vessel clearance, and the use of a preceding phosphate buffered saline washout was not associated with higher vascular clearance scores. The degree of case-to-case variability observed within treatment conditions suggests that procedural factors can play an important role on perfusion quality in addition to perfusate composition.