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Mitochondria–endoplasmic reticulum (ER) contact sites (MERCS) are signaling hubs that are involved in vital cellular processes such as calcium signaling, lipid metabolism, autophagy, and apoptosis. Alterations in MERCS dynamics and composition are closely associated with severe human diseases such as neurodegenerative disorders and cancer. The BCL-2 protein family are primary regulators of mitochondrial apoptosis and key players in maintaining cellular homeostasis that operate at MERCS to fulfill essential cellular functions. Apoptotic machinery-induced cyclic GMP-AMP synthase (cGAS)–STING pathway activation and proinflammatory signaling require mitochondria–ER communication and are affected in cancer, Alzheimer's disease, and Parkinson's disease. Mitochondria–endoplasmic reticulum (ER) contact sites (MERCS) function as transient signaling platforms that regulate essential cellular functions. MERCS are enriched in specific proteins and lipids that connect mitochondria and the ER together and modulate their activities. Dysregulation of MERCS is associated with several human pathologies including Alzheimer's disease (AD), Parkinson's disease (PD), and cancer. BCL-2 family proteins can locate at MERCS and control essential cellular functions such as calcium signaling and autophagy in addition to their role in mitochondrial apoptosis. Moreover, the BCL-2-mediated apoptotic machinery was recently found to trigger cGAS–STING pathway activation and a proinflammatory response, a recognized hallmark of these diseases that requires mitochondria–ER interplay. This review underscores the pivotal role of MERCS in regulating essential cellular functions, focusing on their crosstalk with BCL-2 family proteins, and discusses how their dysregulation is linked to disease. [ABSTRACT FROM AUTHOR]

Uropathogenic strains of E. coli (UPEC) is a leading cause of sepsis, deploying multiple virulence factors to evade host immune responses. Notably, alpha-hemolysin (HlyA) produced by UPEC is implicated in septic symptoms associated with bacteremia, correlating with thrombocytopenia, a critical indicator of organ dysfunction and a predictor of poorer patient prognosis. This study meticulously explores the impact of sublytic concentrations of HlyA on platelets. Findings reveal that HlyA triggers an increase in intracellular calcium, activating calpain and exposing phosphatidylserine to the cell surface, as validated by flow cytometric experiments. Electron microscopy reveals a distinctive balloon-like shape in HlyA-treated platelets, indicative of a procoagulant state. The toxin induces the release of procoagulant extracellular vesicles and the secretion of alpha and dense granules. Overall, the results point to HlyA inducing a necrotic-like procoagulant state in platelets. The effects of sublytic concentrations of HlyA on both erythrocytes and platelets could have a potential impact on capillary microcirculation. Targeting HlyA emerges as a viable therapeutic strategy to mitigate the adverse effects of UPEC infections, especially in South American countries where these infections are endemic, underscoring its significance as a potential therapeutic target. • HlyA induces Ca+2 influx, with the concomitant PS exposure and calpain activation. • HlyA induces PS positive-microvesicles delivery from platelets. • HlyA-induced the secretion of alpha and dense granules. • HlyA-induced the exposure of the integrin P-selectin and the release of ATP. • HlyA induces a necrotic-like procoagulant state in platelets. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening severe inflammatory reaction caused by the host's dysregulated response to infection. Sepsis-induced myocardial dysfunction (SIMD) has been confirmed to occur in 50 % of patients with septic shock. Currently, the pathophysiological mechanism of SIMD is complex, and there is no targeted treatment. Elabela is another endogenous ligand of Aplnr (APJ). The protective effect of APJ on the heart has been proven. Elabela (Ela) has been shown to have a variety of cardiovascular protective effects. However, there are no studies demonstrating the protective effect of Ela-APJ axis on SIMD. In vivo, C57BL/J mice were injected subcutaneously with 1 mg/kg/d Ela for 2 weeks, and in vitro, AC16 cells were treated with 1 μM Ela for 24 h. A 7–0 thread was used to ligate the distal end of the cecum, followed by puncture with a 20-gauge needle. Once a small amount of fluid leaks out, release the cecum back into the abdominal cavity. We measured the survival rates of the mice, performed ultrasound on their hearts, and evaluated the effects of the treatments. The serum and cell supernatant were extracted to detect myocardial injury markers and pyroptosis-related indicators. Western blotting was used to detect autophagy and pyroptosis-related protein. Molecular docking and other experiments were also used to detect changes in related proteins. In vivo, Ela significantly improved the survival rate of septic mice, improved cardiac function, and reduced the production of myocardial injury markers, oxidative stress and pyroptosis. In vitro, Ela unblocked autophagy flow by affecting TFEB transcription. Autophagy reduces inflammation and oxidative stress by selectively degrading inflammatory bodies and ultimately alleviates pyroptosis. We had demonstrated for the first time that in sepsis, Ela promoted the degradation of inflammasomes, reduced oxidative stress, and inhibited the occurrence of pyroptosis by unblocking autophagy flow. Pyroptosis is considered another type of programmed necrosis, which mainly relies on activated caspase-1 to cleave gasdermin D (GSDMD) protein. LPS can activate caspase-1 by activating the NLRP3 inflammasome. Elabela can inhibit oxidative stress, reduce pyroptosis, and ultimately reduce cardiomyocyte damage by regulating the autophagy pathway. [Display omitted] • Elabela improves sepsis-induced myocardial dysfunction. • Elabela can be combined with APJ to activate the Elabela-APJ axis. • Elabela can regulate the intracellular calcium concentration to facilitated the flow of autophagy. • Elabela alleviates myocardial pyroptosis caused by sepsis by regulating autophagy. [ABSTRACT FROM AUTHOR]

Cellular ceramics are highly valued for their exceptional mechanical properties and low density, making them suitable for applications like thermal insulation, impact absorption, and biomedical implants. However, high-temperature sintering required for densification can cause issues like shrinkage, loss of precision, and degradation of functional additives. This work reports a novel approach for fabricating 3D printed cellular ceramics under ambient conditions using CO 2 -cured γ-C 2 S ink via direct ink writing. The printed green body is converted into a dense matrix of CaCO 3 and silica gel upon exposure to a CO 2 -rich environment without high temperature sintering. The non-sintered cellular ceramics achieve high compressive and specific strength comparable to sintered counterparts, with the square-shaped structure exhibiting the best performance of 104 and 171 MPa in terms of in-plane and out-of-plane compressive strength, respectively. Further, the in-situ analysis reveals distinct deformation modes and crack propagation patterns across structures, providing insights into structure-property relationships to guide the design of high-strength non-sintered cellular ceramics. [ABSTRACT FROM AUTHOR]

Sonoporation-based delivery has great promise for noninvasive drug and gene therapy. After short-term membrane resealing, the long-term function recovery of sonoporated cells affects the efficiency and biosafety of sonoporation-based delivery. It is necessary to identify the key early biological signals that influence cell fate and to develop strategies for manipulating the long-term fates of sonoporated cells. Here, we used a customized experimental platform with a single cavitating microbubble induced by a single ultrasound pulse (frequency: 1.5 MHz, pulse length:13.33 μs, peak negative pressure: ∼0.40 MPa) to elicit single-site reversible sonoporation on a single HeLa cell model. We used a living-cell microscopic imaging system to trace the long-term fates of sonoporated HeLa cells in real-time for 48 h. Fluorescence from intracellular propidium iodide and Fluo-4 was used to evaluate the degree of sonoporation and intracellular calcium fluctuation (ICF), respectively. Changes in cell morphology were used to assess the long-term cell fates (i.e., proliferation, arrest, or death). We found that heterogeneously sonoporated cells had different long-term fates. With increasing degree of sonoporation, the probability of normal (proliferation) and abnormal fates (arrest and death) in sonoporated cells decreased and increased, respectively. We identified ICF as an important early event for triggering different long-term fates. Reversibly sonoporated cells exhibited stronger proliferation and restoration at lower extents of ICF. We then regulated ICF dynamics in sonoporated cells using 2-APB or BAPTA treatment to reduce calcium release from intracellular organelles and enhance intracellular calcium clearance, respectively. This significantly enhanced the proliferation and restoration of sonoporated cells and reduced the occurrence of cell-cycle arrest and death. Finally, we found that the long-term fates of sonoporated cells at multiple sites and neighboring cells were also dependent on the extent of ICF, and that 2-APB significantly enhanced their viability and reduced death. Thus, using a single HeLa cell model, we demonstrated that regulating intracellular calcium can effectively enhance the proliferation and restoration capabilities of sonoporated cells, therefore rescuing the long-term viability of sonoporated cells. These findings add to our understanding of the biophysical process of sonoporation and help design new strategies for improving the efficiency and biosafety of sonoporation-based delivery. [Display omitted] [ABSTRACT FROM AUTHOR]

Intracellular calcium dynamics is key to regulating various physiological events. Myotube formation by myoblast fusion is controlled by the release of Ca2+ from the endoplasmic reticulum (ER), and the calpain (CAPN) family is postulated to be an executioner of the process. However, the activation of a specific member of the family or its physiological substrates is unclear. In this study, we explore the involvement of a CAPN in myoblast differentiation. Time-course experiments showed that the reduction in potential substrates of calpains, c-Myc and STAT3 (signal transducer and activator of transcription 3) and generation of STAT3 fragments occurred multiple times at an early stage of myoblast differentiation. Inhibition of the ER Ca2+ release suppressed these phenomena, suggesting that the reduction was dependent on the cleavage by a CAPN. CAPN5 knockdown suppressed the reduction. In vitro reconstitution assay showed Ca2+- and CAPN5-dependent degradation of c-Myc and STAT3. These results suggest the activation of CAPN5 in differentiating myoblasts. Fusion of the Capn5 knockdown myoblast efficiently occurred; however, the upregulation of muscle-specific proteins (myosin and actinin) was suppressed. Myofibrils were poorly formed in the fused cells with a bulge where nuclei formed a cluster, suggesting that the myonuclear positioning was abnormal. STAT3 was hyperactivated in those fused cells, possibly inhibiting the upregulation of muscle-specific proteins necessary for the maturation of myotubes. These results suggest that the CAPN5 activity is essential in myoblast differentiation. [ABSTRACT FROM AUTHOR]

Incidence of mental health disorders are rising in modernity, with psychological stress linked to a propensity for developing various chronic diseases due to a relative inability of the body to counter the allostatic load on cellular level. Despite these high rates of comorbidities associated with posttraumatic stress disorder (PTSD), there is still a lack of understanding in terms of the peripheral effects of PTSD on tissue level. Therefore, the purpose of this study was to profile basal dermal fibroblast functional status in PTSD using a wide range of markers involved in the cell-to-cell communication facilitated by fibroblasts. Primary dermal fibroblasts derived from patients diagnosed with PTSD (n = 11) and matched trauma exposed controls (i.e. who did not develop PTSD, n = 10) were cultured using standard techniques. The patients and controls were matched based on age, sex, body-mass index (BMI) and lifestyle. The growth rate, population doubling time, cell surface marker expression (CD31, FNDC5) (flow cytometry), secretome (TIMP-2, MMP-9) (ELISAs), intracellular signalling capacity (Fluo-4 Ca2+ flux) and gene expression (IL-6, IL-10, PTX-3, iNOS, Arg1) were compared between groups. The data illustrated significant PTSD-associated fibroblast conditioning resulting in a blunted signalling capacity. This observation highlights the importance of including tissue-specific investigations in future studies focused on elucidating the association between PTSD and subsequent risk for somatic disease. • PTSD conditions fibroblasts in the periphery. • Fibroblast signalling capacity is generally blunted in PTSD. • Data suggest PTSD-associated fibroblast desensitization to stimuli. [ABSTRACT FROM AUTHOR]

Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro , the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse. • Different treatments for equine sperm capacitation were evaluated in two media. • Combination of capacitating inducers boosts phosphorylation and acrosomal exocytosis. • Capacitating conditions improve sperm's zona pellucida binding capacity. [ABSTRACT FROM AUTHOR]

Evidence from animal models and human studies clearly demonstrate that sodium-glucose cotransporter-2 (SGLT2) inhibitors have systemic and tissue-specific anti-inflammatory effects that may be independent of glycemic control and weight loss. SGLT2 inhibitors may achieve their anti-inflammatory effects through various mechanisms, including reduction of reactive oxygen species, inflammasome activation, intracellular sodium and calcium levels, circulating uric acid, and mitochondrial dysfunction. The direct and indirect mitigation of inflammatory pathways by SGLT2 inhibitors may partially explain their cardiac and renoprotective effects. However, this remains to be formally demonstrated in human studies. Inflammation plays an essential role and is a common feature in the pathogenesis of many chronic diseases. The exact mechanisms through which sodium-glucose cotransporter-2 (SGLT2) inhibitors achieve their much-acclaimed clinical benefits largely remain unknown. In this review, we detail the systemic and tissue- or organ-specific anti-inflammatory effects of SGLT2 inhibitors using evidence from animal and human studies. We discuss the potential pathways through which SGLT2 inhibitors exert their anti-inflammatory effects, including oxidative stress, mitochondrial, and inflammasome pathways. Finally, we highlight the need for further investigation of the extent of the contribution of the anti-inflammatory effects of SGLT2 inhibition to improvements in cardiometabolic and renal outcomes in clinical studies. [ABSTRACT FROM AUTHOR]

GATA1 is one of the master transcription factors in hematopoietic lineages development which is crucial for megakaryocytic differentiation and maturation. Previous studies have shown that distinct GATA1 variants are associated with varying severities of macrothrombocytopenia and platelet dysfunction. To determine the underlying pathological mechanisms of a novel GATA1 variant (c. 686G > A, p. G229D) in a patient with recurrent traumatic muscle hematomas. Comprehensive phenotypic analysis of the patient platelets was performed. Procoagulant platelet formation and function were detected using flow cytometry assay and thrombin generation test (TGT), respectively. The ANO6 expression was measured by qPCR and western blot. The intracellular supramaximal calcium flux was detected by Fluo-5N fluorescent assay. The patient displayed mild macrothrombocytopenia with defects of platelet granules, aggregation, and integrin αIIbβ3 activation. The percentage of the procoagulant platelet formation of the patient upon the stimulation of thrombin plus collagen was lower than that of the healthy controls (40.9 % vs 49.0 % ± 5.1 %). The patient platelets exhibited a marked reduction of thrombin generation in platelet rich plasma TGT compared to the healthy controls (peak value: ∼70 % of the healthy controls; the endogenous thrombin potential: ∼40 % of the healthy controls). The expression of ANO6 and intracellular calcium flux were impaired, which together with abnormal granules of the patient platelets might contribute to defect of procoagulant platelet function. The G229D variant could lead to a novel platelet phenotype characterized by defective procoagulant platelet formation and function, which extended the range of GATA1 variants associated platelet disorders. • A novel GATA1 variant (p. G229D) closely adjacent to NF was identified in the patient with recurrent traumatic hematomas. • The variant caused the defects of the procoagulant platelet formation and function associated with the bleeding symptom. • Defects of ANO6 expression, intracellular calcium flux and granules might jointly affect the procoagulant platelet activity. [ABSTRACT FROM AUTHOR]

Drug-induced liver injury (DILI) is one of the widespread causes of liver injury and immune system plays important role. Abemaciclib (ABE) is a cyclin-dependent kinase inhibitor used as monotherapy or combination therapy in the treatment of breast cancer. Like other kinase inhibitors, the underlying mechanisms of ABE-induced hepatotoxicity are not completely known yet. In the current study, hepatotoxicity of ABE was evaluated with HepG2/THP-1 co-culture model which has been developed in recent years for the evaluation of DILI potential. Following ABE treatment, oxidative stress, mitochondrial damage, cytokine secretion levels, apoptotic/necrotic cell death were determined. According to our results, ROS production along with GSH depletion was observed in HepG2 cells after ABE treatment. ABE promoted secretion of pro-inflammatory mediators (TNF-α and MCP-1) and declined anti-inflammatory cytokine IL-10 release. Besides, NF K β and JNK1 protein expression levels increased following ABE treatment. ABE enhanced intracellular calcium levels, induced early apoptotic and necrotic cell deaths in HepG2 cells. Furthermore, the changes in some mitochondrial parameters including a reducement in intracellular ATP levels and complex V activity; hyperpolarized mitochondrial membrane potential and enhanced mitochondrial ROS levels were observed, whereas mitochondrial mass did not show any differences after ABE treatments. Therefore, ABE-induced hepatotoxic effects is probably via oxidative stress, inflammatory response and necrotic cell death rather than direct mitochondrial toxicity. In conclusion; the study makes a significant contribution to strengthening the infrastructure we have on in vitro toxicity mechanism evaluations, which are the basis of preclinical toxicity studies. • Abemaciclib (ABE) increased ROS production and caused GSH depletion. • Mitochondrial parameters were altered after ABE treatment. • Inflammatory response was triggered following ABE treatment. • ABE enhanced intracellular Ca2+ level and induced necrotic cell death. [ABSTRACT FROM AUTHOR]

Calcium signaling is an essential mediator of signal-controlling gene expression in most developmental systems. In addition, calcium has established extracellular functions as a structural component of biogenic minerals found in complex tissues. In bacteria, the formation of calcium carbonate structures is associated with complex colony morphology. Genes promoting the formation of biogenic minerals are essential for proper biofilm development and protection against antimicrobial solutes and toxins. Here we review recent findings on the role of calcium and calcium signaling as emerging regulators of biofilm formation in beneficial bacteria, as well as essential mediators of biofilm formation and virulence in human pathogens. The presented analysis concludes that the new understanding of calcium signaling may help to improve the performance of beneficial strains for sustainable agriculture, microbiome manipulation, and sustainable construction. Unraveling the roles of calcium may also promote the development of novel therapies against biofilm infections that target calcium uptake, calcium sensors, and calcium carbonate deposition. Calcium signaling is an essential mediator of gene expression in all developmental systems. Intracellular and extracellular calcium recently emerged as regulators of biofilm formation in bacteria and were characterized in more detail for Pseudomonas aeruginosa and Bacillus subtilis. Calmodulin-like proteins play a fundamental role in calcium-dependent gene expression in P. aeruginosa and B. subtilis. The formation of calcium carbonate extracellular structures is associated with complex colony morphology and altered diffusion in microbial biofilms. A broad range of extracellular matrix polymers, including functional amyloids, eDNA, and exopolysaccharides, interact with calcium and promote calcium carbonate mineralization. The genetic pathways underlying microbial mineralization open pathways to utilize synthetic biology for carbon-neutral or low-carbon biocement for construction. Several Ca2+ regulatory and signaling network components may serve as potential drug targets, including calcium channels and calcium-responsive two-component systems. [ABSTRACT FROM AUTHOR]

We investigated the effects of environment calcium challenge and 1α,25(OH) 2 vitamin D 3 (1,25-D 3) on 45Ca2+ influx in the intestine of zebrafish (ZF). In vitro 45Ca2+ influx was analyzed using intestines from fed and fasted fish. ZF were held in water containing Ca2+ (0.02, 0.7, 2.0 mM) to analyze the ex vivo 45Ca2+ influx in the intestine and for histology. Intestines from fish held in water with Ca2+ were incubated ex vivo to characterize ion channels, receptors, ATPases and ion exchangers that orchestrate 45Ca2+ influx. For in vitro studies, intestines were incubated with antagonists/agonist or inhibitors to study the mechanism of 1,25-D 3 on 45Ca2+ influx. Fasted ZF reached a plateau for 45Ca2+ influx at 30 min. In vivo fish at high Ca2+ stimulated ex vivo 45Ca2+ influx and increased the height of intestinal villi in low calcium. In the normal calcium, 45Ca2+ influx was maintained by the reverse-mode Na+/Ca2+ (NCX) activation, Na+/K+-ATPase pump and sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. However, Ca2+ hyperosmolarity is supported by L-type voltage-dependent calcium channels (L-VDCC), transient receptor potential vanilloid subfamily 1 (TRPV1) and Na+/K+-ATPase activity. The calcium challenge causes morphological alteration and changes the ion type-channels involved in the intestine to maintain hyperosmolarity. 1,25-D 3 stimulates Ca2+ influx in normal osmolarity coordinated by L-VDCC activation and SERCA inhibition to keeps high intracellular calcium in intestine. Our data showed that the adult ZF regulates the calcium challenge (per se osmolarity), independently of the hormonal regulation to maintain the calcium balance through the intestine to support ionic adaptation. • In vivo fish exposure to high Ca2+ stimulates ex vivo 45Ca2+ influx. • Calcium challenge alter ion channel types for homeostasis on intestine. • 1,25-D 3 coordinates Ca2+ influx and from stores to keeps intestinal hyperosmolarity. • In vivo exposure to low Ca2+ increase the height of intestinal villi. [ABSTRACT FROM AUTHOR]

The use of triphenylphosphonium cation (TPP+) linked to phenolic compounds by alkyl chains has a significant relevance as a mitochondrial delivery strategy in biomedicine because it affects mitochondrial bioenergetics in models of noncommunicable diseases such as cancer and cardiovascular-related conditions. Studies indicate that a long alkyl chain (10–12 carbon) increases the mitochondrial accumulation of TPP+-linked drugs. In contrast, other studies show that these compounds are consistently toxic to micromolar concentrations (as observed in platelets). In the present study, we evaluated the in vitro effect of three series of triphenylphosphonium-linked acyl hydroquinones derivates on the metabolism and function of human platelets using 3–9 carbons for the alkyl linker. Those were assessed to determine the role of the length of the alkyl chain linker on platelet toxicity. Human platelets were exposed in vitro to different concentrations (2–40 μM) of every compound; cellular viability, phosphatidylserine exposition, mitochondrial membrane potential (ΔΨm), intracellular calcium release, and intracellular ROS generation were assessed by flow cytometry. An in silico energetic profile was generated with Umbrella sampling molecular dynamics (MD). There was an increase in cytotoxic activity directly related to the length of the acyl chain and lipophilicity, as seen by three techniques, and this was consistent with a decrease in ΔΨm. The in silico energetic profiles point out that the permeability of the mitochondrial membrane may be involved in the cytotoxicity of phosphonium salts. This information may be relevant for the design of new TPP+ -based drugs with a safe cardiovascular profile. [Display omitted] • The toxicity of TPP + -linked compounds on platelets correlates with the chain length and lipophilicity. • Mitochondrial membrane may be involved in the cytotoxicity of phosphonium salts. • Mitochondrial-targeted acylhydroquinones can be considered for promising anti-thrombotic treatments. [ABSTRACT FROM AUTHOR]

Sonodynamic therapy (SDT) has emerged as an encouraging noninvasive technique that uses ultrasound to activate targeted agents to induce antitumor effects for the treatment of glioma. With extensive variation in the types of sonosensitizers, protocols for sonication, and model systems, a comprehensive overview of existing preclinical data on the efficacy of SDT in glioma treatment is warranted. Here, we conduct a systematic review of preclinical and early clinical literature on implementing SDT to treat in vitro and in vivo models of glioma. Our findings suggest that coupling sonosensitizers such as 5-aminolevulinic acid, hematoporphyrin monomethyl ether, and sinoporphyrin sodium with focused ultrasound induces robust cytotoxic activity in tumor cells (in vitro and in vivo). These effects are likely mediated by the oxidative stress induced by reactive oxygen species production, apoptotic signaling cascades, and intracellular calcium overload. Future research is needed to better understand the biochemical and mechanistic properties of SDT, and ongoing trials may help elucidate the clinical feasibility of glioma treatment with optimized sonically activated treatments. [ABSTRACT FROM AUTHOR]

The aim of this study was to validate a novel sperm purification device, the VetCount™ Harvester, for use in bovine in vitro fertilization (IVF). The device's performance was compared to BoviPure™ gradient centrifugation, a commercially available and accepted routine technique. Semen quality parameters were assessed for frozen-thawed semen from six different bulls (n = 6) following sperm purification. For each bull two semen subsamples were purified utilizing BoviPure™ gradient centrifugation and the VetCount™ Harvester, including a third subsample as untreated control. Both treatments significantly increased the proportion of progressively motile sperm cells (84.4 ± 14.1% and 85.1 ± 7.8%, respectively) compared to the untreated semen (41.9 ± 18.8%). BoviPure™ gradient and VetCount™ Harvester selected predominantly viable acrosome intact (VAI) sperm cells with low membrane fluidity and low free intracellular calcium concentration [Ca2+] i (76.5 ± 4.4% and 78.6 ± 6.0%). Normalizing [Ca2+] i of VAI sperm cells (non-treated semen: [Ca2+] i = 1) VetCount™ Harvester purified spermatozoa (0.67 ± 0.10) showed significantly lower [Ca2+] i than BoviPure™ treated sperm (0.84 ± 0.14; P < 0.05). Subsequently, the fertilizing ability of the spermatozoa was evaluated performing a competitive fertilization assay. Sperm cells from both treatment groups were fluorescently labelled using different dyes and added in equal amounts to in vitro matured oocytes. After 18 h co-incubation, the origin of the fertilizing sperm cell was evaluated via fluorescence microscopy. In two bulls, VetCount™ Harvester selected sperm that fertilized significantly more oocytes then BoviPure™ treated sperm, in another bull it was the opposite. For three bulls no difference was observed. We conclude that the VetCount™ Harvester selects a high-quality, fertile sperm fraction from frozen-thawed bull semen. However, some considerations have to be kept in mind for the direct use of the isolated sperm fraction in IVF. • The VetCount™ Harvester makes semen preparation gentle and easy. • A novel sperm separation device that selects high-quality, fertile bull sperm. • MitoTrackers® as a useful tool for competitive fertilization studies. [ABSTRACT FROM AUTHOR]

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in plasma glucose. Pulsatile insulin secretion occurs as a result of glucoseinduced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ reduction, beta-cell dysfunction, and an increased risk of type 2 diabetes. However, the mechanistic effects of ER stress on individual calcium channels are not well understood. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5- triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, we treated INS-1 832/13 cells and primary mouse islets with ER stress inducer tunicamycin (TM). We showed TM treatment increased RyR1 mRNA without affecting RyR2 mRNA and decreased both IP3R1 and IP3R3 mRNA. Furthermore, we found stress reduced ER Ca2+levels, triggered oscillations in cytosolic Ca2+under subthreshold glucose conditions, and increased apoptosis and that these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In addition, we demonstrated silencing RyR1-suppressed TM-induced subthreshold cytosolic Ca2+ oscillations, but silencing RyR2 did not affect these oscillations. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the TM-induced cytosolic Ca2+ oscillations and did not protect beta cells from TM-induced apoptosis although xestosponginC inclusion did prevent ER Ca2+ reduction. Taken together, these results show changes in RyR1 play a critical role in ER stress-induced Ca2+ dysfunction and beta-cell apoptosis. [ABSTRACT FROM AUTHOR]

Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein–coupled receptor opsin highly conserved in verte- brates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we exam- ined Opn5m from diverse species using an aequorin lumines- cence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially acti- vated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential acti- vation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types. [ABSTRACT FROM AUTHOR]

Intracellular calcium signaling is essential for many cellular processes, including store-operated Ca2+ entry (SOCE), which is initiated by stromal interaction molecule 1 (STIM1) detecting endoplasmic reticulum (ER) Ca2+ depletion. STIM1 is also activated by temperature independent of ER Ca2+ depletion. Here we provide evidence, from advanced molecular dynamics simulations, that EF-SAM may act as a true temperature sensor for STIM1, with the prompt and extended unfolding of the hidden EF-hand subdomain (hEF) even at slightly elevated temperatures, exposing a highly conserved hydrophobic Phe108. Our study also suggests an interplay between Ca2+ and temperature sensing, as both, the canonical EF-hand subdomain (cEF) and the hidden EF-hand subdomain (hEF), exhibit much higher thermal stability in the Ca2+-loaded form compared to the Ca2+-free form. The SAM domain, surprisingly, displays high thermal stability compared to the EF-hands and may act as a stabilizer for the latter. We propose a modular architecture for the EF-hand-SAM domain of STIM1 composed of a thermal sensor (hEF), a Ca2+ sensor (cEF), and a stabilizing domain (SAM). Our findings provide important insights into the mechanism of temperature-dependent regulation of STIM1, which has broad implications for understanding the role of temperature in cellular physiology. [ABSTRACT FROM AUTHOR]

It is a known fact that cryopreservation initiates premature capacitation in spermatozoa during the cryopreservation process. Protein tyrosine phosphorylation is a landmark of cascade reaction accountable for capacitation or capacitation-like changes in spermatozoa. Therefore, our hypothesis was to test an inhibitor (H89) that reversibly inhibits the cascade reaction responsible for capacitation during the cryopreservation process but does not hamper normal capacitation and fertilizing ability of sperm. For this, sixteen ejaculates were collected from Murrah buffalo bulls (n = 4). Each ejaculate was divided into four equal aliquots and diluted in an egg yolk-based semen dilutor supplemented with 0, 2, 10, and 30 μM concentrations of H89 and cryopreserved. Interestingly, H89 reduces cholesterol efflux from spermatozoa and protects spermatozoa from membrane damage during the cryopreservation process. H89 did not prevent lipid peroxidation of the sperm membrane. H89 reduced intracellular calcium concentration in spermatozoa in a dose-dependent manner, but tyrosine phosphorylation reduction was observed in the 2 and 10 μM H89 groups. The CTC assay revealed that the percentage of uncapacitated spermatozoa in different treatment groups increases in a dose-dependent manner. In the in vitro capacitation medium, the effect of H89 is abolished and spermatozoa underwent normal capacitation, but H89-treated spermatozoa attached to zona pellucida in large numbers compared to untreated spermatozoa. In conclusion, H89 does not only inhibit tyrosine phosphorylation of spermatozoa but it reduces cholesterol efflux and calcium influx, and ultimately reduces capacitation-like changes during the cryopreservation process. • Study disclosed the new role of H89. • H89 reduced cholesterol efflux from spermatozoa. • H89 restricted the influx of calcium in spermatozoa. • H89 prevented phosphorylation of tyrosine-containing proteins. • Effects of H89 were reversed in vitro capacitation media. [ABSTRACT FROM AUTHOR]

Positive-sense RNA viruses modify intracellular calcium stores, endoplasmic reticulum and Golgi apparatus (Golgi) to generate membranous replication organelles known as viral factories. Viral factories provide a conducive and substantial enclave for essential virus replication via concentrating necessary cellular factors and viral proteins in proximity. Here, we identified the vital role of a broad-spectrum antiviral, peruvoside in limiting the formation of viral factories. Mechanistically, we revealed the pleiotropic cellular effect of Src and PLC kinase signaling via cyclin-dependent kinase 1 signaling leads to Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1) phosphorylation and Golgi vesiculation by peruvoside treatment. The ramification of GBF1 phosphorylation fosters GBF1 deprivation consequentially activating downstream antiviral signaling by dampening viral factories formation. Further investigation showed signaling of ERK1/2 pathway via cyclin-dependent kinase 1 activation leading to GBF1 phosphorylation at Threonine 1337 (T1337). We also showed 100% of protection in peruvoside-treated mouse model with a significant reduction in viral titre and without measurable cytotoxicity in serum. These findings highlight the importance of dissecting the broad-spectrum antiviral therapeutics mechanism and pave the way for consideration of peruvoside, host-directed antivirals for positive-sense RNA virus-mediated disease, in the interim where no vaccine is available. Peruvoside inhibits viral replication via Src/ERK kinase cascade activation of CDK1 and GBF1 phosphorylation. Phosphorylated GFB1 successively causes direct/indirect Golgi vesiculation which curtails viral replication. [Display omitted] [ABSTRACT FROM AUTHOR]

The global use of herbicides accounts for more than 48% of total pesticide usage. Picolinafen is a pyridine carboxylic acid herbicide that is predominantly used to control broadleaf weeds in wheat, barley, corn, and soybeans. Despite its widespread use in agriculture, its toxicity in mammals has rarely been studied. In this study, we first identified the cytotoxic effects of picolinafen on porcine trophectoderm (pTr) and luminal epithelial (pLE) cells, which are involved in the implantation process during early pregnancy. Picolinafen treatment significantly decreased the viability of pTr and pLE cells. Our results demonstrate that picolinafen increased the number of sub-G1 phase cells and early/late apoptosis. In addition, picolinafen disrupted mitochondrial function and resulted in the accumulation of intracellular ROS, leading to a reduction in calcium levels in both the mitochondria and cytoplasm of pTr and pLE cells. Moreover, picolinafen was found to significantly inhibit the migration of pTr. These responses were accompanied by the activation of the MAPK and PI3K signal transduction pathways by picolinafen. Our data suggest that the deleterious effects of picolinafen on the viability and migration of pTr and pLE cells might impair their implantation potential. [Display omitted] • Picolinafen decreases cell viability and alters cell cycle progression in trophectoderm. • Picolinafen causes ROS accumulation and disrupts intracellular calcium regulation. • Picolinafen interferes with mitochondrial integrity in trophectoderm and uterine cells. • Picolinafen alters the transcriptional regulation of genes involved in implantation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Ascorbic acid triggered ROS/Ca2+ dependent necrosis. • Extracellular Fe2+/Fe3+ abrogated the cytotoxicity of ascorbic acid in vitro. • Fe2+/Fe3+ supplement diminished the anti-cancer effects of ascorbic acid in vivo. The anti-cancer effect of high concentrations of ascorbic acid (AA) has been well established while its underlying mechanisms remain unclear. The association between iron and AA has attracted great attention but was still controversial due to the complicated roles of iron in tumors. Our study aims to explore the anti-cancer mechanisms of AA and the interaction between AA and iron in cancer. The MTT and ATP assays were used to evaluate the cytotoxicity of AA. Reactive oxygen species (ROS) generation, calcium (Ca2+), and lipid peroxidation were monitored with flow cytometry. Mitochondrial dysfunction was assessed by mitochondrial membrane potential (MMP) detection with JC-1 or tetramethylrhodamine methyl ester (TMRM) staining. Mitochondrial swelling was monitored with MitoTracker Green probe. FeSO 4 (Fe2+), FeCl 3 (Fe3+), Ferric ammonium citrate (Fe3+), hemin chloride (Fe3+) were used as an iron donor to investigate the effects of iron on AA's anti-tumor activity. The in vivo effects of AA and iron were analyzed in xenograft zebrafish and allograft mouse models. High concentrations of AA exhibited cytotoxicity in a panel of cancer cells. AA triggered ROS-dependent non-apoptotic cell death. AA-induced cell death was essentially mediated by the accumulated intracellular Ca2+, which was partly originated from endoplasmic reticulum (ER). Surprisingly, exogenous iron could significantly reverse AA-induced ROS generation, Ca2+ overloaded, and cell death. Especially, the iron supplements significantly impaired the in vivo anti-tumor activity of AA. Our study elucidated the protective roles of iron in ROS/Ca2+ mediated necrosis triggered by AA both in vitro and in vivo , which might shed novel insight into the anti-cancer mechanisms and provide clinical application strategies for AA in cancer treatment. [ABSTRACT FROM AUTHOR]

In this paper, we present a calcium dynamics model that firmly orchestrates exchanges of calcium flux through intracellular/extracellular sources of calcium to investigate cellular activities and calcium homeostasis. The generalized two-dimensional space-time reaction-diffusion model is formulated and analyzed for a wide range of calcium flux. The analytical technique is applied to solve the model by use of the fractional integral transform and some special functions. The existence and uniqueness of the proposed model are discussed. Finally, the obtained results are presented graphically to show the significance of calcium flux by amalgamating the memory of cells and prove the efficiency of the method. • We developed a calcium dynamics model to investigate the interplay of calcium flux based on a reaction-diffusion equation. • We present the computational technique to derive the model's solution using integral transform and special functions. • The existence and uniqueness of the calcium dynamics model are discussed. • We estimated the concentration profile in the presence/absence of VGCC, ER leak, SERCA pump, NCX, and mitochondrial flux. • The modulation of calcium flux provides a noteworthy effect due to the intermediate memory of cells. [ABSTRACT FROM AUTHOR]

Dopamine (DA) is an intensely studied neuromodulator that has been linked to reward signaling, motivation, and movement production, and has been implicated in a multitude of neuropsychiatric diseases. Despite the established role of DA in health and disease, the molecular, cellular, and circuit mechanisms of DA action in vivo remain poorly understood. New tools for monitoring the activity of DA neurons and extracellular DA in target brain regions have revealed that the patterns of DA release are considerably more complex and diverse than previously thought. Advances in cell-type-specific recording techniques, including intracellular effectors such as calcium and protein kinase A (PKA), are challenging current understanding of the modulatory effects of DA on target cells. The neuromodulator dopamine (DA) is essential for regulating learning, motivation, and movement. Despite its importance, however, the mechanisms by which DA influences the activity of target cells to alter behavior remain poorly understood. In this review, we describe recent methodological advances that are helping to overcome challenges that have historically hindered the field. We discuss how the employment of these methods is shedding light on the complex dynamics of extracellular DA in the brain, as well as how DA signaling alters the electrical, biochemical, and population activity of target neurons in vivo. These developments are generating novel hypotheses about the mechanisms through which DA release modifies behavior. [ABSTRACT FROM AUTHOR]

The coordination electrostatic potential has significantly influenced the binding energy of calcium-peptide coordination complexes. However, its impact on calcium bioavailability is unclear. A calcium-binding peptide (CBP) DEDEQIPSHPPR, purified from soybean yogurt was selected. The study selected tetrapeptide residues of acidic amino acids that play an important role in CBP and designed sixteen tetrapeptides rich in carboxyl oxygen atoms to investigate the influence of electrostatic potential on calcium-peptide binding. The results indicated that DEDE exhibited the strongest binding energy (−375.27 kcal/mol) and binding capacity (464.11 ± 2.14 mg/g) with calcium ions compared to other peptides. Moreover, DEDE also increased intracellular calcium concentration the most, being 2.04 times higher than CaCl 2. Calcium ions coordinated with carboxyl oxygen atoms possessing negative electrostatic potential, which correlated positively with the bioavailability of calcium ions. This electrostatic potential analysis provides a theoretical basis for developing and screening bioactive peptides with high calcium bioavailability. [Display omitted] • The coordinated binding of peptides with calcium was facilitated by negative electrostatic potential. • The steric hindrance provided by the combination of Glu and Asp inhibited calcium binding. • Calcium bioavailability was positively correlated with the coordination binding capacity of peptides and calcium. [ABSTRACT FROM AUTHOR]

This study aimed to determine the effects of cryopreservation on sperm functionality and assess post-thaw sperm quality in yellowtail kingfish (Seriola lalandi). Milt designated for cryopreservation was frozen in a solution containing Cortland® medium +1.3 M Me 2 SO + 0.2 M glucose +2 % BSA in a 1:3 ratio (milt:cryoprotectant). Fresh milt was used as the control. The samples were frozen in 0.5 mL straws using a programmable freezing system. After thawing, the samples were evaluated for motility using Computer-Assisted Sperm Analysis (CASA) with a phase-contrast optical microscope. Additionally, flow cytometry and a multimodal reader were employed to assess various sperm parameters, including plasma membrane integrity (SYBR-14/PI), mitochondrial membrane potential (∆ΨMMit: JC-1), DNA fragmentation (TUNEL), ATP content (CellTiter-Glo®), intracellular calcium levels (Fluo-4 AM), lipid peroxidation (LPO), intracellular superoxide (DHE/SYTOX) and glutathione (GSH/GSSG) production, activities of glutathione peroxidase (GPx), catalase (CAT) and 8-oxoguanine glycosylase (OGG1), and detection of 8-hydroxydeoxyguanosine (8-OHdG). In cryopreserved milt, significant differences in motility (64.14 ± 2.2 %), plasma membrane integrity (61.5 ± 2.7 %), mitochondrial membrane potential (55.4 ± 2.4 %), DNA fragmentation (6.7 ± 1.1 %), ATP content (7.5 ± 1.5 nmol/109 spermatozoa), and intracellular calcium levels (59.8 ± 2.5 nM/109 spermatozoa) were observed compared to fresh milt. Differences were also noted in LPO, O 2 − i , GPx, GSH/GSSG, CAT, 8-OHdG and OGG1 between the cryopreserved and control groups. Additionally, correlations between sperm quality parameters, oxidative stress markers, and enzyme activity in cryopreserved spermatozoa were determined. These results suggest that cryopreservation induces significant alterations derived from oxidative damage in the functionality of yellowtail kingfish spermatozoa, and thus further research is necessary to explore the need for using antioxidants in the cryopreservation process of sperm from this species. • Physiological changes in yellowtail kingfish sperm are associated with metabolic enzymes and ions. • Cryopreservation of Seriola lalandi sperm leads to significant changes in sperm quality parameters. • The results highlight the need for the use of antioxidants in the cryopreservation of kingfish sperm. [ABSTRACT FROM AUTHOR]

Intracellular calcium (Ca2+) is involved in a plethora of cell signalling processes and physiological functions. Increases in Ca2+ concentration are bona fide biomarkers of neuronal activity, reflecting the spike count, timing, frequency, and the intensity of synaptic input. The development of genetically encoded calcium indicators (GECIs) was a significant advancement in modern neuroscience that enabled real-time visualisation of neuronal activity at single-cell resolution. These indicators leverage the conformational changes induced by calcium-binding proteins, such as calmodulin (CaM) or troponin C (TnC). Harnessing protein engineering approaches such as directed evolution yielded new GECIs with enhanced sensitivity, kinetics, and brightness. Notably, the development of calcium-based integrators, such as scFLARE (single-chain fast light- and activity-regulated expression), convert transient raises in cytosolic Ca2+ into a transcriptional readout rather than an optical signal. This review summarises the latest efforts in protein engineering to develop new indicators and molecular systems to sense changes in Ca2+ concentrations. • Overview of genetically encoded calcium indicators (GECIs) based on fluorescent and bioluminescent outputs. • Recent advances in calcium imaging with chemigenetic and ultrasonic reporters. • The summary and description of calcium integrators based on their molecular mechanisms and readouts. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) isolated from tissues such as bone marrow, cord, cord blood, etc., are frequently used as feeder layers to expand hematopoietic stem/ progenitor cells (HSCs/HSPCs) in vitro. They are also co-infused with the HSCs to improve the efficacy of transplantation. However, the MSCs sourced from non-hematopoietic tissues could have suboptimal hematopoiesis-supportive ability. Likewise, the functionality of the MSCs is known to decline after continuous in vitro culture – an unavoidable manipulation to get clinically relevant cell numbers. Hence, it may be necessary to boost the hematopoiesis-supportive ability of the long-term cultured MSCs so that they can, in turn, be used to prime the HSCs before their clinical applications. Here, I show that priming human bone marrow-derived MSCs (BMSCs) with appropriately selected signaling modifiers and integrin-activating bioactive peptides boosts their hematopoiesis-supportive ability, as seen by the formation of a significantly higher number of colonies from the bone marrow-derived mononuclear cells (MNCs) and extensive proliferation of CD34+ HSCS briefly interacted with them. Priming the BMSCs with signaling modifiers is a cost-effective and time-efficient process as synthesizing these small molecule compounds is relatively inexpensive – an advantage in clinical settings. The approach of briefly interacting the donor HSCs/HSPCs with the primed BMSCs just before their infusion into the recipients' bodies could save the cost of long-term ex vivo expansion of HSCs. This concept could also find applications in other regenerative medicine protocols after identifying suitable pharmacological modulators that have the desired effects on the target cells. [Display omitted] [ABSTRACT FROM AUTHOR]

Neutrophils that are overactivated can cause inflammatory diseases. Neutrophils possess various surface receptors, including G-protein-coupled chemoattractant receptors, which assist in recognizing pathogen attacks and the inflammatory environment. Therefore, targeting G-protein-coupled chemoattractant receptors and their downstream molecules is important for preventing abnormal neutrophil activation. This study examines the effects and underlying mechanism of myriscagayanone C, a new compound obtained from the fruit of myristica cagayanensis, on neutrophil respiratory burst induced by fMLP. The immunoblotting assay was conducted to assess the mechanisms by which myriscagayanone C inhibits fMLP-induced respiratory burst by disrupting the translocation of Akt to the cellular membrane. Briefly, myriscagayanone C suppressed the production of superoxide anions induced by fMLP on human neutrophils in a concentration-dependent manner (IC 50 : 4.73 ± 0.68 μM). Myriscagayanone C blocked fMLP-induced Akt translocation to the cell membrane, inhibiting AktT308 and AktS473 phosphorylation by PDK1Y373/376 and mTORS2481, respectively. Myriscagayanone C inhibited fMLP-induced p47phox phosphorylation and translocation. Myriscagayanone C did not inhibit the activity of PI3K, the amount of phosphatidylinositol (3, 4, 5)-trisphosphate, or the translocation of phosphorylated-PDK1Y373/376 and -mTORS2481 to the membrane. Myriscagayanone C did not inhibit fMLP-induced PKC, Src, ERK1/2, p38 phosphorylation, and intracellular calcium mobilization. Myriscagayanone C did not inhibit the chemotaxis and CD11b expression induced by fMLP. Myriscagayanone C did not inhibit PMA-induced superoxide anion production and neutrophil extracellular trap formation. According to this data, myriscagayanone C inhibits fMLP-induced neutrophil superoxide anion production by interrupting the translocation of Akt to the plasma membrane, which affects the NADPH oxidase activity by preventing p47phox phosphorylation and translocation. [Display omitted] • Myriscagayanone C is a new extract from the fruit of myristica cagayanensis. • Myriscagayanone C specifically suppressed superoxide anion production induced by fMLP in human neutrophils without affecting that induced by PMA. • Myriscagayanone C did not affect the activity of PI3K and the translocation of PDK1 and mTOR induced by fMLP. • Myriscagayanone C inhibits fMLP-induced neutrophil superoxide anion production by specifically interrupting the translocation of Akt to the plasma membrane, which affects the NADPH oxidase activity by preventing p47phox phosphorylation and translocation. [ABSTRACT FROM AUTHOR]

Central neurons of the common goldfish (Carassius auratus) are exceptional in their capacity to survive Ca2+-induced excitotoxicity and cell death during hypoxia. Horizontal cells (HCs) are inhibitory interneurons of the retina that are tonically depolarized by the neurotransmitter, glutamate, yet preserve intracellular Ca2+ homeostasis. In HCs isolated from goldfish, and in the absence of glutamatergic input, intracellular Ca2+ concentration ([Ca2+] i) is protected from prolonged exposure to hypoxia by mitochondrial ATP-dependent K+ (mK ATP) channel activity. In the present study, we investigated the effects of hypoxia upon [Ca2+] i in isolated HCs during tonic activation by glutamate to better predict the effects of hypoxia in the active retina. Dynamic changes in [Ca2+] i were measured using the ratiometric Ca2+ indicator, Fura-2. Application of 100 μM glutamate during hypoxia (P O2 = 25 mmHg) produced a 1.3-fold greater rise in [Ca2+] i compared to the same glutamate stimulus during normoxia. The hypoxia-dependent increase in [Ca2+] i was abolished by application of 5-hydroxydecanoic acid, which renders mK ATP channels inactive. Extracellular Ca2+ did not contribute to the elevated [Ca2+] i observed during hypoxia, as the effect persisted in Ca2+-free solution and during application of verapamil, an L-type Ca2+ channel blocker. By contrast, inhibition of the mitochondrial Ca2+ uniporter or ryanodine receptors (with ruthenium red or ryanodine, respectively) abolished the hypoxia-dependent rise in [Ca2+] i. This study reports an mK ATP -dependent rise in [Ca2+] i during hypoxia in HCs activated by glutamate, and suggests roles for the mitochondria and intracellular Ca2+ stores in regulating this mechanism. [Display omitted] • Horizontal cells are inhibitory interneurons of the retina. • In goldfish, hypoxia increases cell calcium when glutamate receptors are activated. • The increased calcium is dependent upon mitochondrial ion channel activity. • In this mechanism, calcium originates from intracellular stores. • This mechanism may support hypoxic tolerance in the goldfish retina. [ABSTRACT FROM AUTHOR]

• Ion channels play an important role in regulating the long-term changes in TMV. • Experimental results are supported by a theoretical model. • Long-term changes in TMV after electroporation are temperature/cell-type dependent. • Monitoring long-term changes in TMV after electroporation remains challenging. Electroporation causes a temporal increase in cell membrane permeability and leads to prolonged changes in transmembrane voltage (TMV) in both excitable and non-excitable cells. However, the mechanisms of these TMV changes remain to be fully elucidated. To this end, we monitored TMV over 30 min after exposing two different cell lines to a single 100 µs electroporation pulse using the FLIPR Membrane Potential dye. In CHO-K1 cells, which express very low levels of endogenous ion channels, membrane depolarization following pulse exposure could be explained by nonselective leak current, which persists until the membrane reseals, enabling the cells to recover their resting TMV. In U-87 MG cells, which express many different ion channels, we unexpectedly observed membrane hyperpolarization following the initial depolarization phase, but only at 33 °C and not at 25 °C. We developed a theoretical model, supported by experiments with ion channel inhibitors, which indicated that hyperpolarization could largely be attributed to the activation of calcium-activated potassium channels. Ion channel activation, coupled with changes in TMV and intracellular calcium, participates in various physiological processes, including cell proliferation, differentiation, migration, and apoptosis. Therefore, our study suggests that ion channels could present a potential target for influencing the biological response after electroporation. [ABSTRACT FROM AUTHOR]

β-asarone, an effective volatile oil component of Acorus chinensis, has been found to hold beneficial effects on Parkinson's disease (PD), but its mechanism remains incompletely understood. Drosophila melanogaster with PTEN induced kinase 1 (PINK1) mutations, a prototype PD model, was used in this study. We found that calcium chelation profoundly alleviated a spectrum of PD symptoms. Whereas, calcium supplementation made the case worse, suggesting accumulated calcium contributes to progression of PD. β-asarone administration decreased Ca2+ level in PD flies, accompanied by alleviated behavioral and neural defects. Further study demonstrated that β-asarone downregulated L-type Ca2+ channels (Dmca1D), which was increased in PD flies. Besides, β-asarone decreased expression of 1,4,5 - trisphosphate receptor (Itpr), which is responsible for calcium release from endoplasmic reticulum (ER). Knockdown of either Dmca1D or Itpr specifically in dopaminergic neurons alleviated behavioral and neural defects in PD flies. While overexpression of Itpr aggravated PD symptoms. The results indicated that increased intracellular calcium influx and release triggers dysregulation of calcium homeostasis in PD flies. And β-asarone prevents PD by restoring Ca2+ homeostasis. Overall, the study demonstrated that β-asarone can serve as a new prospective medication against PD or other diseases associated with dysregulation of Ca2+ homeostasis. [ABSTRACT FROM AUTHOR]

Disruption and dysregulation of cellular calcium channel function can lead to diseases such as ischemic stroke, heart failure, and arrhythmias. Corresponding calcium channel drugs typically require preliminary efficacy evaluations using in vitro models such as cells and simulated tissues before clinical testing. However, traditional detection and evaluation methods often encounter challenges in long-term continuous monitoring and lack calcium specificity. In this study, a dynamic monitoring system based on ion-sensitive membranes for light-addressable potentiometric sensor (LAPS) was developed to meet the demand for monitoring changes in extracellular calcium ion (Ca2+) concentration in live cells. The effects of Ca2+ channel agonists and blockers on 2D and 3D HL-1 cells were investigated, with changes in extracellular Ca2+ concentration reflecting cellular calcium metabolism, facilitating drug evaluation. Additionally, calcium imaging technology with optical addressing capability complemented the LAPS system's ability to perceive 3D cell morphology, enhancing its drug evaluation capabilities. This work provides a novel, label-free, specific, and stable technique for monitoring cellular calcium metabolism. It achieves both continuous monitoring at single points and custom sensing area calcium imaging, holding significant implications for drug screening and disease treatment related to human calcium homeostasis. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), continues to be a major contributor to global mortality rates. To effectively combat this pandemic, TB control has to be enhanced in several areas, including point-of-care diagnostics, shorter and safer drug regimens, and preventative vaccination. The latest findings have highlighted autophagy as a host-defense mechanism that eradicates many invading bacteria, including M. tb. Thus, novel approaches like the stimulation of autophagy using various pharmaceutical drugs can be undertaken to deal with this noxious pathogen. The present study has been formulated to evaluate the anti-mycobacterial potential of Furamidine, a DNA minor groove binder (MGB). Initially, a non-cytotoxic concentration of Furamidine (10 µM) was used to assess its impact on the intracellular persistence of mycobacteria in differentiated THP-1 (dTHP-1) cells. Furamidine treatment compromised intracellular mycobacterial growth compared to control cells. Autophagy, a well-known host-defensive strategy, was investigated as a possible contributor to revealing the mechanism of action. Multiparametric approaches such as LC3-I to II conversion, protein level expression of different autophagic markers, and MDC staining were employed to study autophagic response that conclusively suggested the autophagy induction potential of Furamidine in dTHP-1 cells. Further, elevated LC3-II expression and increased autophagic vacuole accumulation under Baf-A1 treatment demonstrated the positive regulation of autophagic flux upon Furamidine treatment. Mechanistic investigations showed increased intracellular calcium (Ca2+) level expression, SIRT1, pAMPK, and FOXO3a activation upon its treatment. Inhibition of Ca2+ level expression suppressed Ca2+-mediated-FOXO3a level in Furamidine-treated cells. Furthermore, administering various inhibitors hampered the Furamidine-induced autophagy that impacted intracellular mycobacteria clearance. These results conclude that Furamidine triggered the Ca2+/pAMPK/SIRT1/FOXO3a pathway, causing less mycobacterial load in dTHP-1 cells. [Display omitted] • Furamidine induces autophagy to clear intracellular mycobacteria in dTHP-1 cells. • Furamidine treatment enhances intracellular Ca2+ levels in dTHP-1 cells. • Activation of pAMPK by Ca2+ regulates SIRT1-FOXO3a-mediated autophagy upon Furamidine treatment. • Inhibition of Ca2+-pAMPK-SIRT1-FOXO3a abolishes the anti-mycobacterial effect of Furamidine in dTHP-1 cells. [ABSTRACT FROM AUTHOR]

Food-grade titanium dioxide (E171) has been under scrutiny in the last decade since its possible adverse effects; however, the cellular mechanisms underlying E171 toxicity have not been thoroughly described. We aimed to compare the effects of E171 on endoplasmic reticulum (ER) homeostasis in normal and cancer colon cells. We exposed normal, carcinoma, and adenocarcinoma cells to 0.1, 1, 10, 50 and 100 μg/cm2 of E171 for 24, 48 and 72 h, and we evaluated ER stress, cell viability, titanium uptake, intracellular calcium concentration, and gene expression related to unfolded protein response (UPR) and chaperone pathways. Cell viability decreased only after 72 h of exposure to 100 μg/cm2 of E171. Adenocarcinoma cells internalized higher titanium amounts than normal and carcinoma cells, but the effects in ER distribution, intracellular calcium concentration, and gene expression were similar among the three cell lines. The expression of UPR and chaperone pathways were downregulated at the lowest concentrations but upregulated at the highest concentrations in the three cell lines. E171 induces ER stress through alterations in ER distribution, intracellular calcium, and UPR and chaperone protein pathways. [Display omitted] • E171 induces endoplasmic reticulum (ER) stress. • E171 induces no differential toxicity between normal and cancer colon cells. • Normal and cancer colon cells can uptake E171 at a similar extent. • E171 alters ER structure and increases intracellular calcium. • E171 deregulated the UPR and chaperone pathway. [ABSTRACT FROM AUTHOR]

With sessile habits, sponges (phylum Porifera) are susceptible to marine pollution impacts and recently microplastics were identified as one source of contamination. Microplastics have a physical impact on filtration rates and plastics additives such as di(2-ethylhexyl)phthalate (DEHP), a ubiquitous marine contaminant, were already identified in their tissues indicating bioaccumulation. However, few studies assessed the impacts of such compounds in its physiology. One verified effect of phthalate exposure is the arrest of the contraction cycles observed in the sponge Hymeniacidon heliophila. In this work, proteomics of DEHP exposed organisms of this species was performed to identify modifications in signaling pathways that could lead to this arrest and recovery. The results indicate that exposed organisms had different expressed 5HT receptors, associated to intracellular calcium signaling, the principal pathway to contraction animals. The Myosin Light-Chain Kinase (MLCK) pathway is detected only in exposed organisms as well as components linked to binding of organic cyclic compounds. Results show that for healing from DEHP exposure, H. heliophila may activate an alternative contraction signaling pathway, the MLCK pathway. These coordinate mechanisms could restore contractions in H. heliophila after acute exposure to DEHP. Research into the impact of microplastics on organisms uses animal models known to science such as mussels. In our work, we tested the effects of a plastic additive, DEHP, on the physiology of a much less studied marine organism: sponges. [Display omitted] • Sponges (phylum Porifera) are affected by marine pollution, including microplastics, which can disrupt their filtration abilities and accumulate in their tissues. • Di(2-ethylhexyl)phthalate (DEHP), a common marine pollutant, accumulates in sponges and affects their physiology. • DEHP exposure altered the expression of 5HT receptors, linked to intracellular calcium signaling, which is crucial for sponge contractions. • An alternative contraction pathway, the Myosin Light-Chain Kinase (MLCK) pathway, was activated in exposed sponges. • Components associated with binding organic cyclic compounds were also found in exposed organisms. [ABSTRACT FROM AUTHOR]

The use of a video method based on the Digital Image Correlation (DIC) algorithm from experimental mechanics to estimate the displacements, strain field, and sarcolemma length in a beating single-cell cardiomyocyte is proposed in this work. The obtained deformation is then correlated with the calcium signal, from calcium imaging where fluorescent dyes sensitive to calcium Ca 2+ are used. Our proposed video-based method for simultaneous contraction and intracellular calcium analysis results in a low-cost, non-invasive, and label-free method. This technique has shown great advantages in long-term observations because this type of intervention-free measurement neutralizes the possible alteration in the beating cardiomyocyte introduced by other techniques for measuring cell contractility (e.g., Traction Force Microscopy, Atomic Force Microscopy, Microfabrication or Optical tweezers). Three tests were performed with synthetically augmented data from cardiomyocyte images to validate the robustness of the algorithm. First, a simulated rigid translation of a referenced image is applied, then a rotation, and finally a controlled longitudinal deformation of the referenced image, thus simulating a native realistic deformation. Finally, the proposed framework is evaluated with real experimental data. To validate contraction induced by intracellular calcium concentration, this signal is correlated with a new deformation measure proposed in this article, which is independent of cell orientation in the imaging setup. Finally, based on the displacements obtained by the DIC algorithm, the change in sarcolemma length in a contracting cardiomyocyte is calculated and its temporal correlation with the calcium signal is obtained. [ABSTRACT FROM AUTHOR]

[Display omitted] • High intracellular calcium amounts inhibit in vitro T cell proliferation. • tBHQ, a calcium enhancer and NRF2 activator, inhibits T cell proliferation. • tBHQ ameliorated DSS-induced colitis in mice. • High calcium amounts could be a potential target to lower T cell activation. Optimal T cell activation is critical to orchestrate adaptive immune responses. Calcium is critical for T cell activation and integrates signaling pathways necessary to activate key transcription factors. In fact, patients with calcium channelopathies are immunodeficient. Here, we investigated the effects of different concentrations of intracellular calcium on activation of mouse T cells. High intracellular calcium amounts inhibited in vitro T cell proliferation as evidenced by a decreased cell cycling-to-hypodiploidy ratio in two models of activation: the combination of phorbol 12-myristate 13-acetate (PMA) and Ionomycin (an ionophore)/Thapsigargin (a SERCA inhibitor) or plate bound anti-CD3 and anti-CD28. High intracellular calcium amounts increased the production of reactive oxygen species (ROS) in T cells activated with PMA and Ionomycin and scavenging excess ROS using N-acetyl cysteine (NAC) rescued the decrease in cycling-to-hypodiploidy ratio. To test the universality of our observations, we studied the effects of tert -Butylhydroquinone (tBHQ), a SERCA inhibitor and Nrf2 activator. tBHQ alone did not increase intracellular calcium amounts but the intracellular calcium amounts increased when tBHQ was used in combination with PMA. Also, tBHQ inhibited T cell activation in a dose-dependent manner in both in vitro models of T cell activation. Importantly, intraperitoneal injection of tBHQ ameliorated Dextran Sodium Sulfate (DSS)-induced colitis in mice as evidenced by rescue of colon length shortening and lower disease activity index. Overall, this study identifies high calcium amounts as a potential target to lower T cell activation. The implications of these observations are discussed in the context of calcium modulating drugs that are used to treat various diseases. [ABSTRACT FROM AUTHOR]

Nanomedicine for mediating chemotherapy/ion-interference combination cancer therapy. [Display omitted] As an emerging cancer therapeutic modality, ion-interference therapy (IIT), mediated by the elevated levels of intracellular calcium ions (Ca2+), hasgarneredsignificant research attention in the field of cancer theranostics. However, most reported IIT strategies have heavily relied on Ca-based inorganic compounds, such as calcium peroxide and calcium carbonate,whose biosafety remains under investigation due to their high density and uncertainbiodegradability.This study describes a novel strategy for efficiently encapsulating doxorubicin (DOX) andCa2+ using the CaSO 4 gradient method fordrugremote loading. In breast cancer cell models, we demonstrated a synergistic anticancer effect between Ca2+ and DOX, and elucidated the reactive oxygen species(ROS)-based antitumor mechanisms. This research represents the first instance of nanomedicine inducing combined chemotherapy/ion-interference therapy for cancer treatment without the use of insoluble inorganic compounds, thus offering enhanced biosafety. [ABSTRACT FROM AUTHOR]

CIRI causes excitotoxic injury, which is characterized by an increase in VGLUT1/2 expression and a decrease in GLT-1/ GLAST/ ATP1α1 expression, leading to an increase in glutamate release and transport disorders, and ultimately resulting in glutamate excitotoxic injury. Moreover, the expression of glutamate metabolism related proteins was reversed and glutamate levels in synaptic gaps were decreased after the injection of exogenous leptin. Our studies indicate that leptin may be beneficial against CIRI through neuroprotective effects by improving the expression of glutamate transporters and metabolism-related enzymes. [Display omitted] • In this study, the possible protective mechanism of exogenous leptin on CIRI mice was investigated by measuring neurological function scores, areas of cerebral infarction, and pathological damage in the mice, as well as the release and uptake of glutamate. • This study tentatively confirmed that exogenous Leptin significantly reduced the degree of neurological deficits, areas of cerebral infarction, pathological damage changes in brain tissue, and the number of positive cells in degenerate neurons in CIRI mice, resulting in neuroprotective effects. • In this study, it was found that Leptin may reduce the release of Glu by decreasing the expression of VGLUT1, thereby attenuating the excitotoxicity of Glu, but has no significant effect on the expression of VGLUT2. • This study is the first to find that Leptin may alleviate the excitotoxicity of Glu by increasing the expression of GLT-1 and GLAST on AST, thereby increasing Glu transport in synaptic clefts. Ischemic stroke is characterized by high morbidity and mortality and a lack of effective therapeutic interventions. Leptin plays an important role in regulating oxidative stress, angiogenesis, hematopoiesis, etc. Although recent studies have found a neuroprotective effect of leptin, little is known about its role in cerebral ischemia. This study explores the possible roles and potential preventative mechanisms of leptin in cerebral ischemia–reperfusion injury (CIRI). An in vivo middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was used to replicate the CIRI model, low (0.5 mg/kg), medium (1 mg/kg) and high (2 mg/kg) concentrations of leptin were injected intraperitoneally immediately after inserting the embolic line. After 1.5 h of ischemia and 24 h of reperfusion, we examined the neural function of the mice, collected brain tissue for histological examination, and screened for the optimal concentrations of leptin intervention. On this basis, we observed the changes of cortical apoptosis injury, intracellular calcium fluorescence intensity and astrocyte glial fibrillary acidic protein (GFAP) expression and morphological changes. In addition, we also tested the expression of transporters and metabolism-related enzymes (VGLUT-1, VGLUT-2, GLAST, GLT-1, GS, ATP1α1), the expression of inflammatory factors and the content of glutamate (Glu). Compared with the I/R group, we found that leptin improved neurological deficits, reduced the area of infarcts, maintained the normal morphology of astrocytes (AST), downregulated the expression of VGLUT-1, and upregulates the expression of GLT-1 and GLAST, thereby reducing the content of Glu in the synaptic cleft. Our studies suggest that leptin may have a neuroprotective effect by decreasing the excitotoxicity of glutamate. [ABSTRACT FROM AUTHOR]

Transient receptor potential vanilloid 1 (TRPV1) pathway can result in the disruption of intracellular calcium ion (Ca2+) homeostasis, but how to effectively control the process of Ca2+ overload needs to be addressed for tumor treatment. Herein, we developed a mild photothermal activation of TRPV1 pathway strategy for enhanced Ca2+ overload therapy of hepatocellular carcinoma, in which the multifunctional nanoparticle (CNQ) loading with CaCO 3 , a novel polycyclic conjugated small molecule (2Br-NDIA) and quercetin (Qu) as the core for the efficient mild photothermal therapy (PTT). The photothermal property of CNQ can specifically activate the TRPV1 channel, which leads to mitochondrial Ca2+ overloading and the disruption of intracellular Ca2+ homeostasis. This CNQ-mediated activation of the TRPV1 channel by mild PTT provides an emerging paradigm for enhancing mitochondrial Ca2+ overload therapy and dismantling tumor self-defense, safely expanding cancer therapies for highly refractory tumors. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a degenerative disorder characterised by amyloid-beta aggregates activated by the accumulation of lipid molecules and their derivatives, especially 7-ketocholesterol (7KCh), an oxidised lipid that plays a great part in the progression of AD. The current therapeutics need bio-potential molecules and their biomedical application preventing 7KCh-induced cytotoxicity. In this study, bornyl acetate (BA) and menthol (ME), the natural monoterpenes were investigated for their neuroprotective effects against 7KCh-induced SH-SY5Y cells and their effects were compared to the standard drug galantamine (GA). 7KCh-induced changes like lipid accumulation, amyloid generation, free radical generation, acetylcholinesterase levels, calcium accumulation and mitochondrial membrane integrity were analysed in SH-SY5Y cells with or without BA and ME treatment. Furthermore, various mediators involved in the amyloidogenic, inflammatory and apoptotic pathways were studied. In our results, the cells induced with 7KCh upon co-treatment with BA and ME significantly reduced lipid accumulation and amyloid generation through toll-like receptor (TLR) 4 suppression and enhanced ATP binding cassette (ABCA) 1-mediated clearance. Co-treatment with BA and ME concurrently regulated oxidative stress, acetylcholinesterase activity, mitochondrial membrane potential and intracellular calcification altered by 7KCh-induced SH-SY5Y cells. Moreover, 7KCh-induced cells showed elevated mRNA levels of misfolded protein markers and apoptotic mediators which were significantly downregulated by BA and ME co-treatment. In addition, the protein expression of amyloidogenic, proinflammatory as well as pro-apoptotic markers was decreased by BA and ME co-treatment in 7KCh-induced cells. Overall, BA and ME mediated inhibition of amyloidogenic activation and cell survival against 7KCh-induced inflammation, thereby preventing the onset and progression of AD in comparison to GA. [Display omitted] • Bornyl acetate (BA) and Menthol (ME) prevented 7-ketocholesterol accumulation and generation of oxidative stress. • BA and ME ameliorated nuclear damage, mitochondrial and intracellular calcium accumulation. • BA and ME inhibited BACE1-mediated APP processing and Aβ1–42 generation in regulating amyloidogenic signalling pathway. • Anti-inflammatory effect of BA and ME downregulated caspase-3/NF-κB inflammatory mediators preventing neuronal cell death. [ABSTRACT FROM AUTHOR]

Dry eye disease (DED) is caused by inflammation on the ocular surface and insufficient quality or production of the tear film. Due to various harmful environmental conditions, a gradual increase of DED cases has been reported. This study aims for a comprehensive in vitro pharmacological and phytochemical profiling of two different Buddleja officinalis Maxim. extracts to assess their potential for the treatment of DED. A hydroethanolic (BO-HE) and a lead-like enhanced (BO-LLE) B. officinalis extract were phytochemically characterized by UHPLC-UV-MS and UHPLC-ELSD analyses. Afterwards, the effects of either BO-HE or BO-LLE on in vitro dry eye models, including human corneal epithelial cell-transformed (HCE-T) cells, immortalized human meibomian gland epithelial cells (IHMGECs) and human leukemia monocytic (THP-1) cells, and Jurkat cells, were investigated. Both extracts exhibited strong anti-inflammatory properties with free radical scavenging activities and reduction of intracellular reactive oxygen species (ROS) in UVB-exposed HCE-T cells. Treatment with BO-HE or BO-LLE showed wound healing capacities. Moreover, both extracts differentially modulate mediator secretion in UVB-exposed HCE-T cells. In IHMGECs, the size of secreted lipid droplets was larger in BO-LLE treated cells. As for immune cells, a significant reduction in levels of TNF-α and IL-6 secretion by lipopolysaccharide (LPS)-exposed THP-1 cells was observed. Additionally, BO-LLE effectively inhibited intracellular calcium influx in Jurkat cells even at low concentrations. The results of this study demonstrate pharmacological potential of B. officinalis flowers for the treatment of DED pathology with BO-LLE exerting a slightly more beneficial profile than BO-HE. [Display omitted] [ABSTRACT FROM AUTHOR]

We investigated the link between ferroptosis and the miR-223-3p/inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) pathway in diabetic kidney disease (DKD). Blood samples from DKD patients and healthy controls were analysed for iron ions, calcium ions, and lipid peroxidation. High-glucose-induced glomerular endothelial cells were used to simulate DKD. MiR-223-3p overexpression or silencing was achieved using adenoviruses, affecting ferroptosis regulators (glutathione peroxidase 4 [GPX4], cystine/glutamate transporter (xCT), and long-chain acyl-CoA synthetase 4 [ACSL4]) and ITPR3. DKD patients showed elevated levels of iron ions, calcium ions, and lipid peroxidation. High glucose downregulated miR-223-3p, reducing xCT and GPX4 expression and increasing ACSL4 expression. MiR-223-3p was confirmed to target ITPR3 through luciferase reporter assay. MiR-223-3p overexpression reversed high-glucose-induced effects on ferroptosis markers and ITPR3 expression. In summary, high glucose levels decreased miR-223-3p expression, leading to increased calcium ion levels and ferroptosis, potentially through ITPR3 modulation. These findings provide insights into the mechanisms underlying DKD and its potential therapeutic targets. • miR-223-3p in DKD: High-glucose reduces miR-223-3p expression, suggesting its protective role in diabetic kidney disease (DKD). • Ferroptosis in DKD: Elevated iron, calcium, and lipid peroxidation in DKD patient samples highlight ferroptosis's role in DKD. • ITPR3 as miR-223-3p Target: ITPR3 is a confirmed direct target of miR-223-3p, key to understanding its regulatory role in DKD. • High-Glucose Impact: High-glucose lowers GPX4 and xCT while raising ACSL4, promoting ferroptosis in DKD. • Therapeutic Potential: miR-223-3p overexpression reverses high-glucose-induced ferroptosis and ITPR3 dysregulation, suggesting therapeutic use. [ABSTRACT FROM AUTHOR]

LVLPGE is an angiotensin-converting enzyme (ACE) inhibitory peptide extracted from broccoli protein hydrolysates. The antihypertensive mechanism of LVLPGE was explored in the present study. The results indicated that the IC 50 value of LVLPGE was 19.49 μM, and its binding to ACE occurred via a static fluorescence quenching involving the secondary structure changes of ACE. In addition, LVLPGE competitively bound to the active site of ACE through hydrogen bonding. Furthermore, LVLPGE markedly enhanced the levels of the vasodilator nitric oxide (NO) in the Ang I-induced HUVECs model, suppressed the expression of vasoconstrictor endothelin-1 (ET-1), and increased the phosphorylation of eNOS. Moreover, LVLPGE was found to enhance the production of cyclic guanosine monophosphate (cGMP), which leading to a subsequent decrease in intracellular calcium concentration, ultimately facilitating the relaxation of vascular smooth muscles and lowering the blood pressure. These results demonstrated that LVLPGE achieves the antihypertensive effects by inhibiting ACE activity and activating the eNOS/NO/cGMP signaling pathway. This study provides new insights into the antihypertensive mechanism of LVLPGE and contributes to the high-value utilization of broccoli resources. [Display omitted] • LVLPGE is an ACE inhibitory peptide extracted from broccoli protein hydrolysate. • LVLPGE exerted its antihypertensive effects through the eNOS/NO/cGMP signaling pathway. • LVLPGE bound to ACE occurred via a static fluorescence quenching. • LVLPGE competitively bound to the active site of ACE through hydrogen bonding. [ABSTRACT FROM AUTHOR]

Redox signaling mediated by reversible oxidative cysteine thiol modifications is crucial for driving cellular adaptation to dynamic environmental changes, maintaining homeostasis, and ensuring proper function. This is particularly critical in pancreatic β-cells, which are highly metabolically active and play a specialized role in whole organism glucose homeostasis. Glucose stimulation in β-cells triggers signals leading to insulin secretion, including changes in ATP/ADP ratio and intracellular calcium levels. Additionally, lipid metabolism and reactive oxygen species (ROS) signaling are essential for β-cell function and health. We employed IodoTMT isobaric labeling combined with tandem mass spectrometry to elucidate redox signaling pathways in pancreatic β-cells. Glucose stimulation significantly increases ROS levels in β-cells, leading to targeted reversible oxidation of proteins involved in key metabolic pathways such as glycolysis, the tricarboxylic acid (TCA) cycle, pyruvate metabolism, oxidative phosphorylation, protein processing in the endoplasmic reticulum (ER), and insulin secretion. Furthermore, the glucose-induced increase in reversible cysteine oxidation correlates with the presence of other post-translational modifications, including acetylation and phosphorylation. Proper functioning of pancreatic β-cell metabolism relies on fine-tuned regulation, achieved through a sophisticated system of diverse post-translational modifications that modulate protein functions. Our findings demonstrate that glucose induces the production of ROS in pancreatic β-cells, leading to targeted reversible oxidative modifications of proteins. Furthermore, protein activity is modulated by acetylation and phosphorylation, highlighting the complexity of the regulatory mechanisms in β-cell function. [Display omitted] • Glucose-induced redox signaling via reversible cysteine oxidation is vital for β-cell adaptation and homeostasis. • Glucose increases ROS production in β-cells, selectively targeting reversible oxidation of key metabolic proteins. • Reversible cysteine oxidation affects glycolysis, TCA cycle, pyruvate metabolism, and insulin secretion. • Reversible cysteine oxidation correlates with acetylation and phosphorylation, forming a complex regulatory system. • These findings reveal how β-cells adjust metabolism to maintain glucose homeostasis and β-cell health. [ABSTRACT FROM AUTHOR]

Astrocytes, the most abundant type of glial cell, are electrically non-excitable cells that use intracellular calcium (Ca2+) for functional regulation. Changes in intracellular Ca2+ concentration play important roles in the central nervous system (CNS), as they are involved in the release of gliotransmitters and the control of extracellular ion concentrations, thereby affecting the regulation of neuronal excitability, CNS homeostasis, and behavior. Intracellular calcium mobilization in astrocytes is known to be mediated via inositol 1,4,5-trisphosphate receptors (IP 3 Rs), particularly IP 3 R2, and its association with CNS pathogenesis has been widely reported. In addition, the existence of IP 3 R2-independent calcium signaling has recently been postulated; however, the detailed mechanisms and its role in astrocyte functions and CNS pathogenesis are still poorly understood. In this paper, we describe the putative mechanisms underlying IP 3 R1-dependent calcium signaling in astrocytes and its effects on the reactive state, compare this signaling with IP 3 R2-dependent calcium signaling, and discuss its contribution to chronic itch-like behavior. • IP 3 R1-dependent Ca2+ signaling persistently activates STAT3 in astrocytes. • Astrocytic IP 3 R1-dependent Ca2+ responses are small and long-lasting. • Reactive astrocytes induced by IP 3 R1-dependent STAT3 activation underlie chronic itch. • IP 3 R1 and IP 3 R2 of astrocytes differ in the features of Ca2+ responses and function. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB). And the PE_PGRS family members of M. tuberculosis are closely associated with virulence and antigen presentation but with function largely elusive. PE_PGRS1(Rv0109) contained 7 Ca2+ binding domains of GGXGXD/NXUX (X is any amino acid), which can reduce intracellular Ca2+ surge. In addition, PE_PGRS1 can mitigate the activation of PERK branch in endoplasmic reticulum (ER) stress by down-regulating the expression of CHOP, Bip, p-PERK, p-eIF2α, and ATF4. Interestingly, we found that two splicing variations of Bax/Bcl-2, Baxβ, and Bcl-2α, were differentially expressed after infection with Ms_PE_PGRS1, and may be involved in the regulation of apoptosis. Hence, this study identified that PE_PGRS1 is a novel calcium-associated protein that can decrease intracellular Ca2+ levels and the PERK axis. And the weakening of the PERK-eIF2α-ATF4 axis reduces THP-1 macrophages apoptosis, promotes the survival of mycobacteria in macrophages. [Display omitted] • PE_PGRS1 is a novel calcium-associated protein of Mycobacterium tuberculosis. • PE_PGRS1 can reduce intracellular calcium levels. • PE_PGRS1 can mitigate the activation of PERK-eIF2α-ATF4 axis. • PE_PGRS1 differentially regulates the expression of splice variants Baxβ and Bcl-2α. • PE_PGRS1 can increase the survival of mycobacteria in THP-1 macrophages. [ABSTRACT FROM AUTHOR]

Transient receptor potential melastatin (TRPM) channel is involved in cell proliferation and cell survival. Eight members (TRPM1–8) are within the TRPM subfamily. The current study is aimed to investigate TRPM6 expression in human oral carcinogenesis. Sixty-six oral squamous cell carcinomas (OSCCs), 47 oral potentially malignant disorders (OPMD) with moderate-severe epithelial dysplasia (ED), 28 OPMD with mild ED, and 33 normal oral mucosa (NOM) samples were subjected to immunohistochemical staining. Two human oral cancer cell lines (OCCLs), an oral premalignant cell line (DOK), and a normal oral keratinocyte culture (HOK) were used for Western blot analysis. OCCLs were evaluated for proliferation, migration, invasion assays, and intracellular calcium concentration. TRPM6 protein expression in OSCC was significantly increased as compared with normal samples. Protein expression of TRPM6 in OCCLs was significantly higher as compared with HOK. Significant decreases in degrees of proliferation, migration, invasion, and intracellular calcium concentration were noted in OCCLs with TRPM6 siRNA transfection as compared with those without transfection. Significantly increased TRPM6 protein level was noted in OPMD with moderate-severe ED as compared with those with mild ED. Our results implicate that TRPM6 overexpression is potentially related to human oral carcinogenesis. [ABSTRACT FROM AUTHOR]