Effect of alternative oxidase (AOX) expression on mouse cerebral mitochondria bioenergetics.

Alternative oxidase (AOX) is an enzyme that transfers electrons from reduced quinone directly to oxygen without proton translocation. When AOX from Ciona intestinalis is xenotopically expressed in mice, it can substitute the combined electron-transferring activity of mitochondrial complexes III/IV. Here, we used brain mitochondria from AOX-expressing mice with such a chimeric respiratory chain to study respiratory control bioenergetic mechanisms. AOX expression did not compromise the function of the mammalian respiratory chain at physiological conditions, however the complex IV inhibitor cyanide only partially blocked respiration by AOX-containing mitochondria. The relative fraction of cyanide-insensitive respiration increased at lower temperatures, indicative of a temperature-controlled attenuation of mammalian respiratory enzyme activity. As AOX does not translocate protons, the mitochondrial transmembrane potential in AOX-containing mitochondria was more sensitive to cyanide during succinate oxidation than during malate/pyruvate-supported respiration. High concentrations of cyanide fully collapsed membrane potential during oxidation of either succinate or glycerol 3-phosphate, but not during malate/pyruvate-supported respiration. This confirms AOX's electroneutral redox activity and indicates differences in the proton-translocating capacity of dehydrogenases upstream of the ubiquinone pool. Our respiration data refutes previous proposals for quinone partitioning within the supercomplexes of the respiratory chain, instead supporting the concept of a single homogeneous, freely diffusing quinone pool. Respiration with either succinate or glycerol 3-phosphate promotes reverse electron transfer (RET) towards complex I. AOX expression significantly decreased RET-induced ROS generation, with the effect more pronounced at low temperatures. Inhibitor-sensitivity analysis showed that the AOX-induced decrease in H2O2 release is due to the lower contribution of complex I to net ROS production during RET. Overall, our findings provide new insights into the role of temperature as a mechanism to control respiration and highlight the utility of AOX as a genetic tool to characterize both the distinct pathways of oxygen reduction and the role of redox control in RET.

Targeting PAR2-mediated inflammation in osteoarthritis: a comprehensive in vitro evaluation of oleocanthal's potential as a functional food intervention for chondrocyte protection and anti-inflammatory effects.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by chronic inflammation and progressive cartilage degradation, ultimately leading to joint dysfunction and disability. Oleocanthal (OC), a bioactive phenolic compound derived from extra virgin olive oil, has garnered significant attention due to its potent anti-inflammatory properties, which are comparable to those of non-steroidal anti-inflammatory drugs (NSAIDs). This study pioneers the investigation into the effects of OC on the Protease-Activated Receptor-2 (PAR-2) mediated inflammatory pathway in OA, aiming to validate its efficacy as a functional food-based therapeutic intervention. METHODS: To simulate cartilage tissue in vitro, human bone marrow-derived mesenchymal stem cells (BMSCs) were differentiated into chondrocytes. An inflammatory OA-like environment was induced in these chondrocytes using lipopolysaccharide (LPS) to mimic the pathological conditions of OA. The therapeutic effects of OC were evaluated by treating these inflamed chondrocytes with various concentrations of OC. The study focused on assessing key inflammatory markers, catabolic enzymes, and mitochondrial function to elucidate the protective mechanisms of OC. Mitochondrial function, specifically mitochondrial membrane potential (ΔΨm), was assessed using Rhodamine 123 staining, a fluorescent dye that selectively accumulates in active mitochondria. The integrity of ΔΨm serves as an indicator of mitochondrial and bioenergetic function. Additionally, Western blotting was employed to analyze protein expression levels, while real-time polymerase chain reaction (RT-PCR) was used to quantify gene expression of inflammatory cytokines and catabolic enzymes. Flow cytometry was utilized to measure cell viability and apoptosis, providing a comprehensive evaluation of OC's therapeutic effects on chondrocytes. RESULTS: The results demonstrated that OC significantly downregulated PAR-2 expression in a dose-dependent manner, leading to a substantial reduction in pro-inflammatory cytokines, including TNF-α, IL-1ß, and MCP-1. Furthermore, OC attenuated the expression of catabolic markers such as SOX4 and ADAMTS5, which are critically involved in cartilage matrix degradation. Importantly, OC was found to preserve mitochondrial membrane potential (ΔΨm) in chondrocytes subjected to inflammatory stress, as evidenced by Rhodamine 123 staining, indicating a protective effect on cellular bioenergetics. Additionally, OC modulated the Receptor Activator of Nuclear Factor Kappa-Β Ligand (RANKL)/Receptor Activator of Nuclear Factor Kappa-Β (RANK) pathway, suggesting a broader therapeutic action against the multifactorial pathogenesis of OA. CONCLUSIONS: This study is the first to elucidate the modulatory effects of OC on the PAR-2 mediated inflammatory pathway in OA, revealing its potential as a multifaceted therapeutic agent that not only mitigates inflammation but also protects cartilage integrity. The preservation of mitochondrial function and modulation of the RANKL/RANK pathway further underscores OC's comprehensive therapeutic potential in counteracting the complex pathogenesis of OA. These findings position OC as a promising candidate for integration into nutritional interventions aimed at managing OA. However, further research is warranted to fully explore OC's therapeutic potential across different stages of OA and its long-term effects in musculoskeletal disorders.

In isolated brown adipose tissue mitochondria, UCP1 is not essential for - nor involved in - the uncoupling effects of the classical uncouplers FCCP and DNP.

Recent patch-clamp studies of mitoplasts have challenged the traditional view that classical chemical uncoupling (by e.g. FCCP or DNP) is due to the protonophoric property of these substances themselves. These studies instead suggest that in brown-fat mitochondria, FCCP- and DNP-induced uncoupling is mediated through activation of UCP1 (and in other tissues by activation of the adenine nucleotide transporter). These studies thus advocate an entirely new paradigm for the interpretation of standard bioenergetic experiments. To examine whether these patch-clamp results obtained in brown-fat mitoplasts are directly transferable to classical isolated brown-fat mitochondria studies, we investigated the effects of FCCP and DNP in brown-fat mitochondria from wildtype and UCP1 KO mice, comparing the FCCP and DNP effects with those of a fatty acid (oleate), a bona fide activator of UCP1. Whereas the sensitivity of brown-fat mitochondria to oleate was much higher in UCP1-containing than in UCP1 KO mitochondria, there was no difference in sensitivity to FCCP and DNP between these mitochondria, neither in oxygen consumption rate nor in membrane potential studies. Correspondingly, the UCP1-dependent ability of GDP to competitively inhibit activation by oleate was not seen with FCCP and DNP. It would thus be premature to abandon the established bioenergetic interpretation of chemical uncoupler effects in classical isolated brown-fat mitochondria-and probably also generally in this type of mitochondrial study. Understanding the molecular and structural reasons for the different outcomes of mitoplast and mitochondrial studies is a challenging task.

The role and possible mechanism of the ferroptosis-related SLC7A11/GSH/GPX4 pathway in myocardial ischemia-reperfusion injury.

BACKGROUND: Myocardial ischemia-reperfusion injury (MI/RI) is an unavoidable risk event for acute myocardial infarction, with ferroptosis showing close involvement. We investigated the mechanism of MI/RI inducing myocardial injury by inhibiting the ferroptosis-related SLC7A11/glutathione (GSH)/glutathione peroxidase 4 (GPX4) pathway and activating mitophagy. METHODS: A rat MI/RI model was established, with myocardial infarction area and injury assessed by TTC and H&E staining. Rat cardiomyocytes H9C2 were cultured in vitro, followed by hypoxia/reoxygenation (H/R) modeling and the ferroptosis inhibitor lipoxstatin-1 (Lip-1) treatment, or 3-Methyladenine or rapamycin treatment and overexpression plasmid (oe-SLC7A11) transfection during modeling. Cell viability and death were evaluated by CCK-8 and LDH assays. Mitochondrial morphology was observed by transmission electron microscopy. Mitochondrial membrane potential was detected by fluorescence dye JC-1. Levels of inflammatory factors, reactive oxygen species (ROS), Fe2+, malondialdehyde, lipid peroxidation, GPX4 enzyme activity, glutathione reductase, GSH and glutathione disulfide, and SLC7A11, GPX4, LC3II/I and p62 proteins were determined by ELISA kit, related indicator detection kits and Western blot. RESULTS: The ferroptosis-related SLC7A11/GSH/GPX4 pathway was repressed in MI/RI rat myocardial tissues, inducing myocardial injury. H/R affected GSH synthesis and inhibited GPX4 enzyme activity by down-regulating SLC7A11, thus promoting ferroptosis in cardiomyocytes, which was averted by Lip-1. SLC7A11 overexpression improved H/R-induced cardiomyocyte ferroptosis via the GSH/GPX4 pathway. H/R activated mitophagy in cardiomyocytes. Mitophagy inhibition reversed H/R-induced cellular ferroptosis. Mitophagy activation partially averted SLC7A11 overexpression-improved H/R-induced cardiomyocyte ferroptosis. H/R suppressed the ferroptosis-related SLC7A11/GSH/GPX4 pathway by inducing mitophagy, leading to cardiomyocyte injury. CONCLUSIONS: Increased ROS under H/R conditions triggered cardiomyocyte injury by inducing mitophagy to suppress the ferroptosis-related SLC7A11/GSH/GPX4 signaling pathway activation.

Mitochondrial toxicity of selected natural compounds: in vitro assessment and in silico molecular docking and dynamics simulation.

Prangos uechtritzii Boiss & Hausskn stands out for its rich bioactive constituents including prantschimgin (PRA), imperatorin (IMP), suberosin (SUB), adicardin (ADI), and oxypeucedanin hydrate (OPH) in the Apiaceae family. Although these molecules contribute to several biological activities, their mitochondrial toxicity were not illuminated in depth with the appropriate in vitro and in silico models. Cell viability studies investigated the cytotoxic activities of molecules in HepG2 cells by replacing glucose with galactose due to Warburg effects. Mitochondrial toxicity (mitotoxicity) parameters such as cellular adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) levels were assessed with cytotoxic concentrations of selected molecules. Molecular docking and dynamics studies were also conducted against mitochondrial electron transport chain (ETC) complexes (I-V) with selected compounds. In vitro results showed that PRA, SUB, and IMP reduced cell viability more in galactose media compared to high glucose media in a dose-dependent manner. PRA, IMP, and SUB decreased ATP levels and MMP, especially in the galactose medium. The in silico study revealed that PRA, IMP, and SUB might bind to complexes I-V at different levels. The docking study demonstrated that PRA had the highest binding potential with the complexes, higher than the standard ligands in some cases. The molecular dynamics (MD) simulation study showed that PRA formed stable complexes with complexes II, III, and IV. In addition, PRA was anticipated to remain inside the binding site of complex II most stably during the 230 ns simulation period. Our study suggests that PRA, IMP, and SUB exhibit mitotoxicity.

Dietary alpha-linolenic acid supplementation enhances semen quality, antioxidant capacity, and sperm survival in aging breeder roosters.

Aging in breeder roosters is often accompanied by a decline in semen quality, negatively impacting reproductive performance. This study aimed to investigate the effect of dietary alpha-linolenic acid (ALA), an essential omega-3 polyunsaturated fatty acid, on semen quality, antioxidant capacity, and sperm survival in aging breeder roosters. Roosters were divided into 4 groups and fed diets supplemented with 0%, 0.5%, 1%, and 2% ALA for 6 wk. Results indicated significant improvements in semen volume, sperm viability, and sperm density in ALA-supplemented groups compared to the control (P < 0.05). The 1% ALA group exhibited the most notable enhancements in sperm viability and density. Additionally, ALA supplementation increased the activities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and reduced malondialdehyde (MDA) levels, indicating enhanced antioxidant capacity (P < 0.05). Furthermore, ALA improved mitochondrial membrane potential (MMP) and reduced early and late sperm apoptosis, with the 2% ALA group showing the highest MMP and the lowest ROS-positive rate (P < 0.05). These findings suggest that dietary ALA supplementation enhances semen quality and antioxidant defenses, and mitigates oxidative stress, thus supporting the reproductive health of aging breeder roosters. This study underscores the potential of ALA as a dietary strategy to improve reproductive efficiency in poultry production.

Mitochondrial Dysfunction Associated with mtDNA Mutation: Mitochondrial Genome Editing in Atherosclerosis Research.

BACKGROUND: Atherosclerosis is a complex cardiovascular disease often associated with mitochondrial dysfunction, which can lead to various cellular and metabolic abnormalities. Within the mitochondrial genome, specific mutations have been implicated in contributing to mitochondrial dysfunction. Atherosclerosis-associated m.15059G>A mutation has been of particular interest due to its potential role in altering mitochondrial function and cellular health. OBJECTIVE: This study aims to investigate the role of the atherosclerosis-associated m.15059G>A mutation in the development of mitochondrial dysfunction in monocyte-- like cells. METHODS: Monocyte-like cytoplasmic hybrid cell line TC-HSMAM1, which contains the m.15059G>A mutation in mtDNA, was used. The MitoCas9 vector was utilized to eliminate mtDNA copies carrying the m.15059G>A mutation from TC-HSMAM1 cybrids. Mitochondrial membrane potential, generation of reactive oxygen species, and lipid peroxidation levels were assessed using flow cytometry. Cellular reduced glutathione levels were assessed using the confocal microscopy. The oxygen consumption rate was measured using polarographic oxygen respirometry. RESULTS: The elimination of the m.15059G>A mutation resulted in a significant increase in mitochondrial membrane potential and improved mitochondrial efficiency while also causing a decrease in the generation of reactive oxygen species, lipid peroxidation, as well as cellular bioenergetic parameters, such as proton leak and non-mitochondrial oxygen consumption. At the same time, no changes were found in the intracellular antioxidant system after the mitochondrial genome editing. CONCLUSIONS: The presence of the m.15059G>A mutation contributes to mitochondrial dysfunction by reducing mitochondrial membrane potential, increasing the generation of reactive oxygen species and lipid peroxidation, and altering mitochondrial bioenergetics. Elimination of the mtDNA containing atherogenic mutation leads to an improvement in mitochondrial function.

Deletion of PMP3 increases ketoconazole resistance by affecting plasma membrane potential in Candida albicans.

Ketoconazole is a classical antifungal drug commonly used in the clinic. With the increased use of ketoconazole in recent years, an increasing number of drug-resistant strains have emerged during clinical treatment. It is well known that fungi acquire drug resistance in multiple ways, while the molecular mechanisms underlying ketoconazole resistance remain for comprehensive exploration. In this study, we found that the expression of the small plasma membrane protein-encoding gene PMP3 was significantly down-regulated in several clinically isolated ketoconazole-resistant strains, indicating the relationship between PMP3 expression and ketoconazole resistance. By knocking out the PMP3, we found that the absence of the Pmp3 resulted in a significant increase in resistance of Candida albicans to ketoconazole, which was also confirmed in a systemic infection model in mice. We further demonstrated that various physiological properties, such as cell membrane fluidity, plasma membrane potential, permeability and ergosterol distribution were altered in the pmp3Δ/Δ mutant, which is associated with the enhanced cellular resistance to ketoconazole. In addition, overexpression rather than deletion of PMP3 alters the hyphal development and biofilm formation capacity in C. albicans. This study reveals the contribution of Pmp3 to alteration of drug resistance in fungal pathogens, which may guide the development of novel antifungal strategies.

The Effect of Calcium Ions on Resting Membrane Potential.

Regulating membrane potential is key to cellular function. For many animal cells, resting membrane potential is predominantly driven by a family of K2P (two-pore domain) potassium channels. These channels are commonly referred to as leak channels, as their presence results in the membrane being permeable to K+ ions. These channels, along with various pumps and exchangers, keep the cell resting membrane potential (Rp) relatively close to potassium's equilibrium potential (EK); however, in many cells, the resting membrane potential is more depolarized than the EK due to a small Na+ ion leak. Raising [Ca2+]O (extracellular Ca2+ concentration) can result in hyperpolarization of the membrane potential from the resting state. The mechanism for this hyperpolarization likely lies in the blockage of a Na+ leak channel (NALCN) and/or voltage-gated Na+ channels. The effects may also be connected to calcium-activated potassium channels. Using Drosophila melanogaster, we here illustrate that changing [Ca2+]O from 0.5 to 3 mM hyperpolarizes the muscle. Replacing NaCl with LiCl or choline chloride still led to hyperpolarization when increasing [Ca2+]O. Replacing CaCl2 with BaCl2 results in depolarization. K2P channel overexpression in the larval muscle greatly reduces the effects of [Ca2+]O on cell membrane potential, likely because potential is heavily driven by the EK in these muscles. These experiments provide an understanding of the mechanisms behind neuronal hypo-excitability during hypercalcemia, as well as the effects of altered expression of K2P channels on membrane potential.

Hyperglycemia adversely affects critical physiological events related to rat sperm capacitation.

Hyperglycemia, the hallmark of diabetes mellitus (DM), is the main cause of DM-related systemic complications, including reproductive issues. Furthermore, the incidence of DM in males of reproductive ages is becoming an increasing concern, as the complexity of sperm capacitation (an essential process for fertilizing the egg) extends beyond conventional sperm parameters such as count, viability, and motility. Capacitation defects cause male infertility, and DM-related hyperglycemia may affect this process. We explore the effects of uncontrolled hyperglycemia on sperm using alloxan-induced hyperglycemic Wistar rats. In addition to assessing conventional sperm parameters, we also evaluated functional indicators, including hyperactivation (HA) with a pharmacological approach and assessed its effects with a computer-assisted sperm analysis (CASA); fluorescence indicators to monitor membrane potential (EmR, DiSC3(5)) and mitochondrial membrane potential (Ψ, JC-1); CatSper activity, using its ability to permeate Na+ ions, and ATP levels with the luciferin-luciferase reaction. We confirmed previous findings with our hyperglycemic model, which replicated the typical reduction on conventional sperm parameters. In sperm from hyperglycemic rats, we observed increased motility and HA levels after pharmacological treatment. Additionally, CatSper activity was unaffected by hyperglycemia, while EmR was hyperpolarized under non-capacitating condition. Finally, we noted a low percentage of hyperpolarized Ψ and reduced ATP content. This study highlights the significance of impact of hyperglycemia on sperm physiology and capacitation. We proposed that low ATP levels perturb energy state, signaling pathways, ion channels activity, motility, and HA. Our findings offer insight into DM-associated infertility and potential treatment strategies.

Sulforaphane regulates cell proliferation and induces apoptotic cell death mediated by ROS-cell cycle arrest in pancreatic cancer cells.

Background: Pancreatic cancer (PC), sometimes referred to as pancreatic ductal adenocarcinoma (PDAC), is a major cause of global mortality from cancer. Pancreatic cancer is a very aggressive and devastating kind of cancer, characterized by limited options for therapy and low possibilities of survival. Sulforaphane (SFN), a naturally occurring sulfur-containing compound, is believed to possess anti-inflammatory, anti-obesity, and anti-cancer characteristics. Objective: However, efficient preventative and treatment measures are essential and SFN has been studied for its ability to suppress pancreatic cancer cell proliferation and induce apoptosis. Methods: Here, SFN induced cytotoxicity and apoptosis in PDAC cell lines such as MIA PaCa-2 and PANC-1 cells, as evaluated by cytotoxicity, colony formation, western blot analysis, fluorescence-activated cell sorting (FACS), reactive oxygen species (ROS) detection, caspase-3 activity assay, immunofluorescence assay, and mitochondrial membrane potential assay. Results: In MIA PaCa-2 and PANC-1 cells, SFN inhibited cell survival and proliferation in a dose-dependent manner. The activation of caspase zymogens results in cleaved PARP and cleaved caspase-3, which is associated with an accumulation in the sub G1 phase. Furthermore, SFN increased ROS level and γH2A.X expression while decreasing mitochondrial membrane potential (ΔΨm). Notably, the ROS scavenger N-Acetyl-L-cysteine (NAC) was shown to reverse SFN-induced cytotoxicity and ROS level. Subsequently, SFN-induced cell cycle arrest and apoptosis induction as a Trojan horse to eliminate pancreatic cancer cells via ROS-mediated pathways were used to inhibit pancreatic cancer cells. Conclusion: Collectively, our data demonstrates that SFN-induced cell death follows the apoptosis pathway, making it a viable target for therapeutic interventions against pancreatic cancer.

Modulating voltage-gated sodium channels to enhance differentiation and sensitize glioblastoma cells to chemotherapy.

BACKGROUND: Glioblastoma (GBM) stands as the most prevalent and aggressive form of adult gliomas. Despite the implementation of intensive therapeutic approaches involving surgery, radiation, and chemotherapy, Glioblastoma Stem Cells contribute to tumor recurrence and poor prognosis. The induction of Glioblastoma Stem Cells differentiation by manipulating the transcriptional machinery has emerged as a promising strategy for GBM treatment. Here, we explored an innovative approach by investigating the role of the depolarized resting membrane potential (RMP) observed in patient-derived GBM sphereforming cell (GSCs), which allows them to maintain a stemness profile when they reside in the G0 phase of the cell cycle. METHODS: We conducted molecular biology and electrophysiological experiments, both in vitro and in vivo, to examine the functional expression of the voltage-gated sodium channel (Nav) in GSCs, particularly focusing on its cell cycle-dependent functional expression. Nav activity was pharmacologically manipulated, and its effects on GSCs behavior were assessed by live imaging cell cycle analysis, self-renewal assays, and chemosensitivity assays. Mechanistic insights into the role of Nav in regulating GBM stemness were investigated through pathway analysis in vitro and through tumor proliferation assay in vivo. RESULTS: We demonstrated that Nav is functionally expressed by GSCs mainly during the G0 phase of the cell cycle, suggesting its pivotal role in modulating the RMP. The pharmacological blockade of Nav made GBM cells more susceptible to temozolomide (TMZ), a standard drug for this type of tumor, by inducing cell cycle re-entry from G0 phase to G1/S transition. Additionally, inhibition of Nav substantially influenced the self-renewal and multipotency features of GSCs, concomitantly enhancing their degree of differentiation. Finally, our data suggested that Nav positively regulates GBM stemness by depolarizing the RMP and suppressing the ERK signaling pathway. Of note, in vivo proliferation assessment confirmed the increased susceptibility to TMZ following pharmacological blockade of Nav. CONCLUSIONS: This insight positions Nav as a promising prognostic biomarker and therapeutic target for GBM patients, particularly in conjunction with temozolomide treatment.

Biomolecular condensates regulate cellular electrochemical equilibria.

Control of the electrochemical environment in living cells is typically attributed to ion channels. Here, we show that the formation of biomolecular condensates can modulate the electrochemical environment in bacterial cells, which affects cellular processes globally. Condensate formation generates an electric potential gradient, which directly affects the electrochemical properties of a cell, including cytoplasmic pH and membrane potential. Condensate formation also amplifies cell-cell variability of their electrochemical properties due to passive environmental effect. The modulation of the electrochemical equilibria further controls cell-environment interactions, thus directly influencing bacterial survival under antibiotic stress. The condensate-mediated shift in intracellular electrochemical equilibria drives a change of the global gene expression profile. Our work reveals the biochemical functions of condensates, which extend beyond the functions of biomolecules driving and participating in condensate formation, and uncovers a role of condensates in regulating global cellular physiology.

Inner Mitochondrial Membrane Peptidase 2-Like Deletion Aggravates Mitochondrial Apoptosis and Inhibits Autophagy After Hyperglycemia Stroke.

This study investigated the effects of inner mitochondrial membrane peptidase 2-like (Immp2l) deletion on mitochondrial apoptosis and mitochondrial autophagy under hyperglycemic conditions. The middle cerebral artery occlusion (MCAO) model was established in wild-type (WT) mice and Immp2l+/- mice; animals were then exposed to hyperglycemic (induced using 1% streptozotocin) and normoglycemic conditions. Tissues were collected at various time points post-reperfusion. The production of reactive oxygen species (ROS) was assessed by fluorescent measurements, and mitochondrial membrane potential was evaluated using a JC-1 assay kit. Autophagy was analyzed by measuring LC3II/LC3I protein expression and Beclin 1 expression. Mitochondrial ultrastructure was examined through transmission electron microscopy (TEM); neuronal autophagosomes were also assessed. Immp2l mutation in a hyperglycemic environment exacerbated brain injury by increasing ROS production, compromising mitochondrial membrane potential, inducing apoptotic cascades, and impairing mitochondrial autophagy. These findings highlight the critical role of Immp2l in modulating the response to hyperglycemic cerebral ischemia-reperfusion (I/R) injury. Furthermore, the deficiency of Immp2l appears to contribute to increased oxidative stress, mitochondrial dysfunction, and cell death, thereby exacerbating brain injury. These data may provide new insights into therapeutic strategies for reducing the impact of diabetes on stroke outcomes.

Toxicity of Dibutyl phthalate (DBP) toward isolated human blood lymphocytes: Apoptosis initiated from intracellular calcium enhancement and mitochondrial/lysosomal cross talk.

Dibutyl phthalate (DBP) is a phthalate ester with wide application in industrial products, so human exposure can happen in workplaces and environment. Conflicting results have been acquired in researches which measured the influences of phthalates contact on immune responses in laboratory animals. Nevertheless, the straight influence of DBP on human lymphocytes and entire mechanisms of its effect against these cells continue to be unexplored. The major purpose of present research was to evaluate the mechanisms which lead to the DBP toxicity on human lymphocytes using accelerated cytotoxicity mechanisms screening (ACMS) technique. Cell viability was determined following12h incubation of lymphocytes with 0.05-1 mM DBP, and mechanistic parameters were assessed after 2, 4 and 6 h of lymphocyte treatment with ½ the IC5012h (0.3 mM), the IC5012h (0.6 mM) and twice the IC5012h (1.2 mM) of DBP. The IC5012 h of a chemical/toxicant is defined as concentration that kills 50 % of cells after 12 h of exposure. The results indicate that DBP exerts toxic effects on isolated human lymphocytes, probably through mitochondrial and lysosomal damage induced by glutathione depletion and oxidative stress. In this study, suppression of cytokines (IL2, INF-gamma and TNF-alpha) production and increase in intracellular calcium were also related to DBP induced lymphocyte toxicity.

SpQuant-SNN: ultra-low precision membrane potential with sparse activations unlock the potential of on-device spiking neural networks applications.

Spiking neural networks (SNNs) have received increasing attention due to their high biological plausibility and energy efficiency. The binary spike-based information propagation enables efficient sparse computation in event-based and static computer vision applications. However, the weight precision and especially the membrane potential precision remain as high-precision values (e.g., 32 bits) in state-of-the-art SNN algorithms. Each neuron in an SNN stores the membrane potential over time and typically updates its value in every time step. Such frequent read/write operations of high-precision membrane potential incur storage and memory access overhead in SNNs, which undermines the SNNs' compatibility with resource-constrained hardware. To resolve this inefficiency, prior works have explored the time step reduction and low-precision representation of membrane potential at a limited scale and reported significant accuracy drops. Furthermore, while recent advances in on-device AI present pruning and quantization optimization with different architectures and datasets, simultaneous pruning with quantization is highly under-explored in SNNs. In this work, we present SpQuant-SNN, a fully-quantized spiking neural network with ultra-low precision weights, membrane potential, and high spatial-channel sparsity, enabling the end-to-end low precision with significantly reduced operations on SNN. First, we propose an integer-only quantization scheme for the membrane potential with a stacked surrogate gradient function, a simple-yet-effective method that enables the smooth learning process of quantized SNN training. Second, we implement spatial-channel pruning with membrane potential prior, toward reducing the layer-wise computational complexity, and floating-point operations (FLOPs) in SNNs. Finally, to further improve the accuracy of low-precision and sparse SNN, we propose a self-adaptive learnable potential threshold for SNN training. Equipped with high biological adaptiveness, minimal computations, and memory utilization, SpQuant-SNN achieves state-of-the-art performance across multiple SNN models for both event-based and static image datasets, including both image classification and object detection tasks. The proposed SpQuant-SNN achieved up to 13× memory reduction and >4.7× FLOPs reduction with < 1.8% accuracy degradation for both classification and object detection tasks, compared to the SOTA baseline.

4-Methylthiazole triggers apoptosis and mitochondrial disruption in HL-60 cells.

BACKGROUND: Thiazole derivatives are gaining prominence in cancer research due to their potent anti-cancer effects and multifaceted biological activities. In leukemia research, these compounds are particularly studied for their ability to induce apoptosis, disrupt mitochondrial membrane potential (MMP), and modulate cell signaling pathways. METHODS AND RESULTS: This study investigates the efficacy of 4-Methylthiazole in inducing apoptosis in HL-60 leukemia cells. Apoptosis was quantified via flow cytometry using FITC Annexin V and propidium iodide staining. Mitochondrial disruption was evaluated through alterations in mitochondrial membrane potential (MMP) as measured by the JC-1 assay. The compound significantly disrupted MMP, activated Caspase-3, and induced the release of Cytochrome C, all of which are critical markers of apoptosis (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). Additionally, treatment with 4-Methylthiazole markedly reduced CD45 and CD123 surface markers, indicating significant phenotypic alterations in leukemia cells (****p < 0.0001). High-dose treatment with 4-Methylthiazole significantly increased ROS levels, suggesting elevated oxidative stress and the presence of intracellular free radicals, contributing to its cytotoxic effects (*p < 0.05). A significant rise in TNF-α levels was observed post-treatment, indicating a pro-inflammatory response that may further inhibit leukemia cell viability. While IL-6 levels remained unchanged, a dose-dependent decrease in IL-10 levels was noted, suggesting a reduction in immunosuppressive conditions within the tumor microenvironment (*p < 0.05). CONCLUSIONS: Overall, 4-Methylthiazole targets leukemia cells through multiple apoptotic mechanisms and modifies the immune landscape of the tumor microenvironment, enhancing its therapeutic potential. This study highlights the need for further clinical investigation to fully exploit the potential of thiazole derivatives in leukemia treatment.

Clustered ARPE-19 cells distinct in mitochondrial membrane potential may play a pivotal role in cell differentiation.

Age-related macular degeneration (AMD) is associated with the dysfunction and degeneration of retinal pigment epithelium (RPE) cells. Here, we examined how the formation and expansions of cell clusters are regulated by the differentiation of the RPE　cells. In this study, ARPE-19 cells were cultivated in standard or differentiation media, i.e., without or with nicotinamide, to evaluate the spreading of cell clusters specified with differentiated cell phenotypes. Mitochondria membrane potential (MMP) and the distribution of the RPE cell clusters was also monitored with or without rotenone, a mitochondrial electron transport chain (ETC) complex I inhibitor. Cultured ARPE-19 cells generated scattered cell clusters composed mostly of smaller size cells expressing the differentiation markers mouse anti-cellular retinaldehyde-binding protein (CRALBP) and Bestrophin only in differentiation medium. After the increase of the number of clusters, the clusters appeared to paracellularly merge, resulting in expansion of the area occupied by the clusters. Of note, the cells within the clusters selectively had high MMP and were in accordance with the expression of RPE differentiation markers. Rotenone repressed the formation of the clusters and decreased intracellular MMP. The above results suggest that clustering of RPE cells with functional mitochondria plays a pivotal role in RPE cell differentiation process and the ETC complex I inhibition greatly influences the composition of RPE cells that are degenerated or differentiation disposed.

Visualizing Mitochondrial Membrane Potential with FRET Probes: Integrating Fluorescence Intensity Ratio and Lifetime Imaging.

Mitochondrial membrane potential (MMP) is crucial for mitochondrial function and serves as a key indicator of cellular health and metabolic activity. Traditional lipophilic cationic fluorescence intensity probes are unavoidably influenced by probe concentration, laser intensity, and photobleaching, limiting their accuracy. To address these issues, we designed and synthesized a pair of fluorescence molecules, OR-C8 and SiR-BA, based on the Förster Resonance Energy Transfer (FRET) mechanism, for dual-modality visualization of MMP. OR-C8 anchors to the inner mitochondrial membrane through strong hydrophobic interactions, while SiR-BA is expelled from mitochondria when MMP decreases, thereby regulating the FRET process. During MMP reduction, the fluorescence intensity and lifetime of OR-C8 increase, while the fluorescence intensity of SiR-BA decreases. By combining changes in fluorescence intensity ratio and fluorescence lifetime, dual-modality visualization of MMP was achieved. This method not only accurately reflects MMP changes but also provides a novel tool for in-depth studies of mitochondrial function and related disease mechanisms, offering significant potential for advancing mitochondrial research and therapeutic development.

Resting trabecular meshwork cells experience constitutive cation influx.

A quintessential sentinel of cell health, the membrane potential in nonexcitable cells integrates biochemical and biomechanical inputs, determines the driving force for ionic currents activated by input signals and plays critical functions in cellular differentiation, signaling, and pathology. The identity and properties of ion channels that subserve the resting potential in trabecular meshwork (TM) cells is poorly understood, which impairs our understanding of intraocular pressure regulation in healthy and diseased eyes. Here, we identified a powerful cationic conductance that subserves the TM resting potential. It disappears following Na+ removal or substitution with choline or NMDG+, is insensitive to TTX, verapamil, phenamil methanesulfonate, amiloride and GsMTx4, is substituted by Li+ and Cs+, and inhibited by Gd3+ and Ruthenium Red. Constitutive cation influx is thus not mediated by voltage-operated Na+, Ca2+, epithelial Na+ (ENaC) channels, Piezo channels or Na+/H+ exchange but may involve TRP-like channels. Transcriptional analysis detected expression of many TRP genes, with the transcriptome pool dominated by TRPC1 followed by expression of TRPV1, TRPC3, TRPV4 and TRPC5. Pyr3 and Pico1,4,5 did not affect the standing current whereas SKF96365 promoted rather than suppressed, Na+ influx. SEA-0400 induced a modest hyperpolarization, indicating residual contribution from Na+/Ca2+ exchange. The resting membrane potential in human TM cells is thus maintained by a constitutive monovalent cation leak current with properties not unlike those of TRP channels. This conductance is likely to influence conventional outflow by setting the homeostatic steady-state and by regulating the magnitude of pressure-induced currents in normotensive and hypertensive eyes.