To search for safe and efficient anti-fatigue active molecules, 16 capsaicin (CAP) derivatives were synthesized by replacing the unsaturated carbon-carbon double bond in capsaicin with a rigid benzene ring via condensation, chlorination, and amidation reactions using vanillylamine hydrochloride as the starting material, with yields ranging from 44.1 to 79.1%. Their structures were confirmed by 1H-NMR, 13C-NMR, and MS (electrospray ionization [ESI]). In vitro assays demonstrated that N8 exerted superior transient receptor potential vanilloid 1 (TRPV1) agonistic activity compared to CAP at a concentration of 10 μM, upregulated peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) expression in a concentration-dependent manner (1.25-10 μM), and showed no significant toxicity to C2C12 myotube cells at 0.78-100 μM. In vivo evaluations in mice (15 mg/kg, 31-d gavage) demonstrated that N8 had no adverse effect on body weight but significantly prolonged rotarod duration (143.5%, p < 0.001) and forced swimming time (75.6%, p < 0.001), increased serum lactate dehydrogenase (LDH) levels (p < 0.01), decreased serum urea nitrogen (SUN) levels (p < 0.001) and lactic acid (LA) accumulation (p < 0.001), and elevated hepatic and muscle glycogen contents (p < 0.001) compared with the fatigue control group. Mechanistic studies via Western blot, mitochondrial fluorescence staining, cellular thermal shift assay, and molecular docking revealed that N8 had better binding stability to TRPV1 than CAP (relative binding rate at 65°C: 86.7 vs. 21.5%), activated the TRPV1 channel, synergistically upregulated the expression of cluster of differentiation 36 (CD36), carnitine palmitoyltransferase 1M (CPT1M), SURF1, and cytochrome c1 (CYC1), promoted mitochondrial biogenesis, and optimized muscle energy metabolism. These results indicate that N8 demonstrates superior anti-fatigue activity both in vitro and in vivo compared to CAP, making it a potential candidate for anti-fatigue drug development.

Cofilin is a central regulator of actin filament turnover, traditionally thought to act through phosphorylation-dependent control of filament assembly. However, its mitochondrial functions remain poorly understood. Here we show that N-terminal α-amino SUMOylation, rather than phosphorylation or actin interaction, governs cofilin-1 translocation to mitochondria and activation of the apoptotic pathway. This modification strengthens the association of cofilin-1 with the mitochondrial import receptors Tom20 and Tom70 through the molecular chaperone HSP70, enabling its delivery to the mitochondrial matrix. Once imported, SUMO-modified cofilin-1 binds cytochrome c1, promotes the dissociation of cytochrome c from complex III, and initiates mitochondrial-mediated apoptosis. These findings redefine cofilin-1 as a regulator of mitochondrial integrity independent of its actin-related roles, uncovering a mechanism by which SUMOylation directs protein targeting and apoptotic signaling. This work broadens current understanding of mitochondrial regulation and may inform therapeutic strategies for diseases linked to defective cell death.

Codelivery of chemo- and protein drugs enhances efficacy but faces challenges from physicochemical differences and inefficient release. We fabricated dual-responsive and tumor-targeting nucleic acid base-inspired nanogels (DTNANGs) for the in vivo codelivery of gemcitabine (Gem) and cytochrome C (CC). The nanogels were engineered through pH-sensitive base-pairing cross-linking between guanine- and cytosine-modified hyaluronic acid derivatives. DTNANGs achieved high drug-loading efficiency (96.3% for Gem, 94.5% for CC). In vitro and in vivo experiments confirmed that DTNANGs demonstrated superior CD44-mediated tumor-targeting capacity, enhanced tumor accumulation, improved cellular uptake, and fast endolysosomal escape. Additionally, CC-loaded DTNANGs increased cell apoptosis by 75.5% in A549 cells, outperforming monotherapy. Moreover, in vivo evaluations in A549 tumor-bearing mice revealed that they significantly inhibited tumor growth (70% inhibitory rate) compared to other control groups, while exhibiting negligible systemic toxicity. Our study lays the groundwork for controlled, effective codelivery of chemo- and protein drugs, enabling precision cancer therapy.

Cytochrome c-mediated mitochondrial apoptosis activation underpins THz wave-induced melanoma cell death.

Pro-apoptotic and mitochondria-disrupting effects of 4-methylthiazole in K562 leukemia cells: A mechanistic investigation.

Impact of IL-17a on Apoptosis and Mucinosis-related Molecules in the Microenvironment of Colorectal Cancer.

Tumor-targeting and redox-responsive photo-cross-linked nanogel derived from multifunctional hyaluronic acid-lipoic acid conjugates for enhanced in vivo protein delivery.

TSGA10, a multifunctional protein critical for mitochondrial coupling and metabolic regulation, plays a paradoxical role in cancer progression and carcinogenesis. Here, we outline a potential mechanism by which TSGA10 mediates metabolism in oncogenesis and thermal modulation. Initially identified in spermatogenesis, TSGA10 interacts with mitochondrial Complex III: it directly binds cytochrome c1 (CytC1). In our model, TSGA10 optimizes electron transport to minimize reactive oxygen species (ROS) and heat production while enhancing Adenosine Triphosphate (ATP) synthesis. In cancer, TSGA10's expression is context-dependent: Its downregulation in tumors like glioblastoma might disrupt mitochondrial coupling, promoting electron leakage, ROS accumulation, and genomic instability. This dysfunction would be predicted to contribute to a glycolytic shift, facilitating tumor survival under hypoxia. Conversely, TSGA10 overexpression in certain cancers suppresses HIF-1α, inhibiting glycolysis and metastasis. TSGA10 and HIF-1α engage in mutual counter-regulation-TSGA10 represses HIF-1α to sustain oxidative phosphorylation (OXPHOS), while HIF-1α suppression of TSGA10 under hypoxia or thermal stress amplifies glycolytic dependency. This interplay is pivotal in tumors adapting to microenvironmental stressors, such as cold-induced mitochondrial uncoupling, which mimics brown adipose tissue thermogenesis to reduce ROS and sustain proliferation. Tissue-specific TSGA10 expression further modulates cancer susceptibility: high levels in the testes and brain may protect against thermal and oxidative damage, whereas low expression in the liver permits HIF-1α-driven metabolic plasticity. Altogether, our model suggests that TSGA10 plays a central role in mitochondrial fidelity. We suggest that its crosstalk with oncogenic pathways position it as a metabolic rheostat, whose dysregulation fosters tumorigenesis through ROS-mediated mutagenesis, metabolic reprogramming, and microenvironmental remodeling. Targeting the hypothesized TSGA10-mediated mitochondrial coupling may offer therapeutic potential to disrupt cancer's adaptive energetics and restore metabolic homeostasis.

ABSTRACTThe mitochondrial bc1 complex catalyzes the oxidation of ubiquinol by reducing cytochrome c. Cytochrome b, the catalytic core of bc1, generates superoxide during the oxidation of ubiquinol. Excessive superoxide production is known to accelerate aging and neurodegeneration. Songbirds (oscine, Passeri) exhibit lower production of mitochondrial reactive oxygen species (ROS) and greatly accelerated evolution of cytochrome b, relative to all other modern birds, suggesting adaptive selection for lower generation of ROS. Here, we identified songbird-specific substitutions in modern bird's cytochrome b amino-acid sequences and examined the high-resolution structures of the chicken bc1 complex in an effort to predict the effect of these substitutions on the function of bc1. Many of the songbird-specific substitutions cluster around sites that are critical for the function of bc1. One cluster of substitutions interacts with heme BH. A second cluster of substitutions interacts with residues in the ubiquinone reduction site, Qi. Both groups of substitution may affect the rate of reduction of ubiquinone at the Qi site. Another cluster of cytochrome b substitutions interacts with the hinge region of the Rieske protein that transfers electron from cytochrome b to cytochrome c1. These songbird-specific substitutions appear to be selected to modulate the rate of both ubiquinol oxidation at the Qo site and ubiquinone reduction at the Qi site thereby modulating the rate of superoxide production. These findings are compatible with the hypothesis that cytochrome b evolution in songbirds was driven by selection of substitutions that reduce the rate of superoxide production thereby increasing songbird lifespan and cognitive abilities.

Smilax glabra roxb. alleviates cisplatin-induced acute kidney injury in mice by activating the Nrf2/HO-1 Signalling Pathway.

SIRT5 mediated succinylation of SUCLA2 regulates TCA cycle dysfunction and mitochondrial damage in pancreatic acinar cells in acute pancreatitis

The relationship between exercise and mitochondrial function is unclear. This study investigated the relationship between voluntary exercise and mitochondrial dynamics in ischemic stroke model mice. This experiment used 54 male C57BL/6 J mice to assess the therapeutic effect of voluntary exercise on ischemic stroke in a middle cerebral artery occlusion (MCAO) model. Body weight and the number of wheel turns were recorded to monitor the physiological condition of the mice. The degree of brain injury was evaluated via hematoxylin and eosin (H&E) staining and measurement of the cerebral infarction volume. Western blotting and immunofluorescence were used to measure dynein-1-like protein 1 (DRP1), mitochondrial fission protein 1 (FIS1), and optic atrophy type 1 (OPA1) levels to assess mitochondrial dynamics and analyze the degree of mitochondrial apoptosis by measuring cytochrome c (CYT-C), cleaved caspase-3, and caspase-3 expression. Voluntary exercise positively affected the behavioral score and infarct volume. H&E staining revealed that voluntary exercise reversed MCAO-induced cortical damage. Furthermore, voluntary exercise improved mitochondrial dynamics by inhibiting DRP1 and FIS1 expression and inducing OPA1 expression. Additionally, the mitochondrial apoptosis pathway was inhibited by down-regulating the expression of CYT-C, cleaved caspase-3, and caspase-3. Voluntary exercise exerts a significant neuroprotective effect against MCAO-induced brain injury by regulating mitochondrial dynamics and the mitochondrial apoptotic pathway.

Mitochondrial holocytochrome c synthase (HCCS) is an essential protein in assembling cytochrome c (cyt c) of the electron transport system. HCCS binds heme and covalently attaches the two vinyls of heme to two cysteine thiols of the cyt c CXXCH motif. Human HCCS recognizes both cyt c and cytochrome c1 of complex III (cytochrome bc1). HCCS is mutated in some human diseases and it has been investigated recombinantly by mutational, biochemical, and reconstitution studies in the past decade. Here, we employ structural prediction programs (e.g., AlphaFold 3) on HCCS and its two substrates, heme and cytochrome c. The results, when combined with spectroscopic and functional analyses of HCCS and variants, provide insights into the structural basis for heme binding, apocyt c binding, covalent attachment, and release of the holocyt c product. Results from in vitro reconstitution of purified human HCCS using cyt c and cyt c1 peptides as acceptors are consistent with the structural modeling of substrate binding. Reconstitution of HCCS and cyt c1 provides an approach to studying cyt c1 assembly, which has been refractile to recombinant in vivo reconstitution (unlike HCCS and cyt c). We propose a structural basis for release of the holocyt c product from HCCS based on in vitro studies and on cryoEM structures of the bacterial cyt c synthase (CcsBA) active site. We analyze the kinetoplastid mitochondrial synthase (KCCS), and hypothesize a molecular evolutionary path from mitochondrial endosymbiosis to the current HCCS.

Intracellular protein delivery has the potential to revolutionize cell-biological research and medicinal therapy, with broad applications in bioimaging, disease treatment, and genome editing. Herein, we demonstrate successful delivery of a functional protein, cytochrome c (CYC), by using a boron cluster anion as molecular carrier of the superchaotropic anion type (B12Br11OPr2-). CYC was delivered into lipid bilayer vesicles as well as living cells, with a cellular uptake ratio approaching 90%. Mechanistic studies showed that CYC was internalized into cells through a permeation pathway directly into the cytoplasm, bypassing endosomal entrapment. Upon carrier-assisted internalization, CYC retained its bioactivity, as reflected by an induced cell apoptosis rate of 25% at low dose (1 µM). This study furbishes a direct protein delivery method by a molecular carrier with high efficiency, confirming the potential of inorganic cluster ions as protein transport vehicles with an extensive range of future cell-biological or biomedical applications.

This study aimed to examine the effects of albumin administration on ischemia-reperfusion in the rat ovary by using biochemical, histological, and immunohistochemical methods. Thirty-two Wistar albino rats were used in the study, and they were divided into 4 groups: control, albumin, placebo, and ischemia-reperfusion. Healthy ovaries were taken from the first group. In the other three groups, 2-hour ischemia and 2-hour reperfusion were applied to the bilateral ovaries. In the albumin group, intraperitoneal albumin (2.5 g/kg, 20% human albumin) was administered 30 minutes before reperfusion, and in the placebo group, the same volume of intraperitoneal saline was administered instead of albumin 30 minutes before reperfusion. Ovarian damage scores, cytochrome C-1 immunoreactivity, total oxidant status, total antioxidant status, and oxidative stress index levels were evaluated. In the statistical analysis performed between the groups, it was seen that the results of the control group were significantly lower than the ischemia-reperfusion group in terms of total oxidant status values (p=0.001), and the results of the ischemia-reperfusion group were significantly higher than the control and albumin groups in terms of oxidative stress index values (p&amp;lt;0.001 and p=0.004, respectively). In histological examinations, the total damage score obtained by evaluating follicular degeneration, edema, vascular congestion, and hemorrhage was found to be significantly higher in the ischemia-reperfusion group than in the control group (p=0.003). According to the immunohistochemical examination results, cytochrome C-1 immunoreactivity in the ischemia-reperfusion group was significantly stronger than the control and albumin groups (p&amp;lt;0.001). We think that albumin administration reduces cytochrome C-1, reactive oxygen species, and oxidative stress levels, therefore it will play a helpful role in the ischemia-reperfusion treatment process.

Data suggest malfunctioning mitochondria reduce oxidation and adenosine triphosphate (ATP) production, disrupting insulin signalling. Cytochrome c(CC), acylcarnitine (AC) and citrate synthase (CS) are essential components of the mitochondria machinery and can be used as reliable biomarkers of mitochondrial dysfunction. This study aimed to determine whether mitochondrial biomarkers (AC, CS and CC) are altered in individuals with type 2 diabetes mellitus (T2DM) and to examine the association between these biomarkers and insulin resistance. A cross-sectional observational study that recruited 170 participants (88 with T2DM and 82 without DM) was conducted. Blood samples were collected from the recruits and analysed for levels of fasting glucose (FBG), AC, CS, CC, insulin, total cholesterol, triglycerides (TG), glycated haemoglobin (HbA1c) and magnesium. Blood pressure (BP) and anthropometric characteristics of participants were also taken. Appropriate formulas were used to determine %body fat, body mass index (BMI), waist-to-hip ratio (WHR), the homeostatic model assessment for insulin resistance (HOMA-IR) and insulin sensitivity (HOMA-β). Patients with T2DM had higher levels of CC, %body fat, FBG, TG, HbA1c, BMI and HOMA-IR than controls (p < 0.05, respectively). Results showed a significant relationship between circulating CC levels versus HOMA-β (r = -0.40, p = 0.001), CS (r = -0.70, p = 0.001) and AC (r = -0.72, p = 0.001) levels in patients with T2DM. The adjusted odds increased in the T2DM patients for VLDL (OR = 6.66, p = 0.002), HbA1c (OR = 6.50, p = 0.001), FPG (OR = 3.17, p = 0.001), TG (OR = 2.36, p = 0.010), being female (OR = 2.09, p = 0.020) and CC (OR = 1.14, p = 0.016). Overall, alterations in mitochondrial biomarkers, measured by AC, CC and CS, were observed in people with T2DM and showed a direct relationship with insulin resistance. These findings are potentially significant in Africa, although additional confirmation from a larger cohort is necessary.

Hepatocellular carcinoma (HCC) presents a persistent challenge to conventional therapeutic approaches. SLC12A5 is implicated in an oncogenic capacity and facilitates the progression of cancer. The objective of this investigation is to scrutinize the inhibitory effects of borax on endoplasmic reticulum (ER)-stress and apoptosis mediated by SLC12A5 in HepG2 cells. Initially, we evaluated the cytotoxic impact of borax on both HL-7702 and HepG2 cell lines. Subsequently, the effects of borax on cellular morphology and the cell cycle of these lines were examined. Following this, we explored the impact of borax treatment on the mRNA and protein expression levels of SLC12A5, C/EBP homologous protein (CHOP), glucose-regulated protein-78 (GRP78), activating transcription factor-6 (ATF6), caspase-3 (CASP3), and cytochrome c (CYC) in these cellular populations. The determined IC50 value of borax for HL-7702 cells was 40.8 mM, whereas for HepG2 cells, this value was 22.6 mM. The concentrations of IC50 (22.6 mM) and IC75 (45.7 mM) of borax in HepG2 cells did not manifest morphological aberrations in HL-7702 cells. Conversely, these concentrations in HepG2 cells induced observable morphological and nuclear abnormalities, resulting in cell cycle arrest in the G1/G0 phase. Additionally, the levels of SLC12A5, ATF6, CHOP, GRP78, CASP3, and CYC were elevated in HepG2 cells in comparison to HL-7702 cells. Moreover, SLC12A5 levels decreased following borax treatment in HepG2 cells, whereas ATF6, CHOP, GRP78, CASP3, and CYC levels exhibited a significant increase. In conclusion, our data highlight the potential therapeutic effects of borax through the regulation of ER stress in HCC by targeting SLC12A5.

Near-infrared light-activated smart nanogels for remotely controlled cytochrome c release and photodynamic therapy

‘Inner mitochondrial membrane peptidase 2 like’ (IMMP2L) is a nuclear-encoded mitochondrial peptidase that has been conserved through evolutionary history, as has its target enzyme, ‘mitochondrial glycerol phosphate dehydrogenase 2′ (GPD2). IMMP2L is known to cleave the mitochondrial transit peptide from GPD2 and another nuclear-encoded mitochondrial respiratory-related protein, cytochrome C1 (CYC1). However, it is not known whether IMMP2L peptidase activates or alters the activity or respiratory-related functions of GPD2 or CYC1. Previous investigations found compelling evidence of behavioural change in the Immp2lKD−/− KO mouse, and in this study, EchoMRI analysis found that the organs of the Immp2lKD−/− KO mouse were smaller and that the KO mouse had significantly less lean mass and overall body weight compared with wildtype littermates (p < 0.05). Moreover, all organs analysed from the Immp2lKD−/− KO had lower relative levels of mitochondrial reactive oxygen species (mitoROS). The kidneys of the Immp2lKD−/− KO mouse displayed the greatest decrease in mitoROS levels that were over 50% less compared with wildtype litter mates. Mitochondrial respiration was also lowest in the kidney of the Immp2lKD−/− KO mouse compared with other tissues when using succinate as the respiratory substrate, whereas respiration was similar to the wildtype when glutamate was used as the substrate. When glycerol-3-phosphate (G3P) was used as the substrate for Gpd2, we observed ~20% and ~7% respective decreases in respiration in female and male Immp2lKD−/− KO mice over time. Together, these findings indicate that the respiratory-related functions of mGpd2 and Cyc1 have been compromised to different degrees in different tissues and genders of the Immp2lKD−/− KO mouse. Structural analyses using AlphaFold2-Multimer further predicted that the interaction between Cyc1 and mitochondrial-encoded cytochrome b (Cyb) in Complex III had been altered, as had the homodimeric structure of the mGpd2 enzyme within the inner mitochondrial membrane of the Immp2lKD−/− KO mouse. mGpd2 functions as an integral component of the glycerol phosphate shuttle (GPS), which positively regulates both mitochondrial respiration and glycolysis. Interestingly, we found that nonmitochondrial respiration (NMR) was also dramatically lowered in the Immp2lKD−/− KO mouse. Primary mouse embryonic fibroblast (MEF) cell lines derived from the Immp2lKD−/− KO mouse displayed a ~27% decrease in total respiration, comprising a ~50% decrease in NMR and a ~12% decrease in total mitochondrial respiration, where the latter was consistent with the cumulative decreases in substrate-specific mediated mitochondrial respiration reported here. This study is the first to report the role of Immp2l in enhancing Gpd2 structure and function, mitochondrial respiration, nonmitochondrial respiration, organ size and homeostasis.

To tackle the obstacles related to tumor targeting and overcome the limitations of single treatment models, we have developed a nanoplatform that is both tumor-targeted and enzyme-responsive. This nanoplatform integrates photothermal gold nanorods (AuNRs) and protein drugs into a single system. This nanosystem, known as AuNRs@HA-mPEG-Deta-LA, was fabricated by modifying gold nanorods (AuNRs) with a polymeric ligand called hyaluronic acid-grafted-(mPEG/diethylenetriamine-conjugated-lipoic acid). The purpose of this fabrication was to load cytochrome c (CC) and utilize it for the synergetic protein-photothermal therapy of cancer. The resulting nanoplatform exhibited a high efficiency in loading proteins and demonstrated excellent stability in different biological environments. Additionally, CC-loaded AuNRs@HA-mPEG-Deta-LA not only enabled localized hyperthermia for photothermal therapy (PTT) with laser irradiation but also facilitated the release of CC under the action of hyaluronidase, an enzyme known to be overexpressed in tumor cells. The confocal imaging results demonstrated that the presence of a specific polymeric ligand on this nanoparticle enhances the internalization of CD44-positive cancer cells, accelerates endo/lysosomal escape, and facilitates the controlled release of CC within the cells. Furthermore, the results of the MTT assay also showed that AuNRs@HA-mPEG-Deta-LA as a protein nanocarrier demonstrated excellent biocompatibility. Importantly, this synergistic therapeutic strategy effectively induced apoptosis in A549 cancer cells by increasing the intracellular concentration of CC and utilizing the photothermal conversion of AuNRs, which was observed to be more effective compared to using only protein therapy or PTT. Therefore, this study showcased a nanoplatform based on AuNRs that has great potential for tumor-targeted protein delivery in combination with PTT in cancer treatment.