2-Aryl-Benzoimidazoles as Type II NADH Dehydrogenase Inhibitors of Mycobacterium tuberculosis.

The nonproton pumping type II NADH dehydrogenase in Mycobacterium tuberculosis is essential for meeting the energy needs in terms of ATP under normal aerobic and stressful hypoxic environmental states. Type II NADH dehydrogenase conduits electrons into the electron transport chain in Mycobacterium tuberculosis, which results in ATP synthesis. Therefore, the inhibition of NDH-2 ensures the abolishment of the entire ATP synthesis machinery. Also, type II NADH dehydrogenase is absent in the mammalian genome, thus making it a potential target for antituberculosis drug discovery. Herein, we have screened a commercially available library of drug-like molecules and have identified a hit having a benzimidazole core moiety (6, H37Rv mc26230; minimum inhibitory concentration (MIC) = 16 µg/mL and ATP IC50 = 0.23 µg/mL) interfering with the oxidative phosphorylation pathway. Extensive medicinal chemistry optimization resulted in analogue 8, with MIC = 4 µg/mL and ATP IC50 = 0.05 µg/mL against the H37Rv mc26230 strain of Mycobacterium tuberculosis. Compounds 6 and 8 were found to be active against mono- and multidrug-resistant mycobacterium strains and demonstrated a bactericidal response. The Peredox-mCherry experiment and identification of single-nucleotide polymorphisms in mutants of CBR-5992 (a known type II NADH dehydrogenase inhibitor) were used to confirm the molecules as inhibitors of the type II NADH dehydrogenase enzyme. The safety index >10 for the test active molecules revealed the safety of test molecules.

Data mining of PubChem bioassay records reveals diverse OXPHOS inhibitory chemotypes as potential therapeutic agents against ovarian cancer.

Focused screening on target-prioritized compound sets can be an efficient alternative to high throughput screening (HTS). For most biomolecular targets, compound prioritization models depend on prior screening data or a target structure. For phenotypic or multi-protein pathway targets, it may not be clear which public assay records provide relevant data. The question also arises as to whether data collected from disparate assays might be usefully consolidated. Here, we report on the development and application of a data mining pipeline to examine these issues. To illustrate, we focus on identifying inhibitors of oxidative phosphorylation, a druggable metabolic process in epithelial ovarian tumors. The pipeline compiled 8415 available OXPHOS-related bioassays in the PubChem data repository involving 312,093 unique compound records. Application of PubChem assay activity annotations, PAINS (Pan Assay Interference Compounds), and Lipinski-like bioavailability filters yields 1852 putative OXPHOS-active compounds that fall into 464 clusters. These chemotypes are diverse but have relatively high hydrophobicity and molecular weight but lower complexity and drug-likeness. These chemotypes show a high abundance of bicyclic ring systems and oxygen containing functional groups including ketones, allylic oxides (alpha/beta unsaturated carbonyls), hydroxyl groups, and ethers. In contrast, amide and primary amine functional groups have a notably lower than random prevalence. UMAP representation of the chemical space shows strong divergence in the regions occupied by OXPHOS-inactive and -active compounds. Of the six compounds selected for biological testing, 4 showed statistically significant inhibition of electron transport in bioenergetics assays. Two of these four compounds, lacidipine and esbiothrin, increased in intracellular oxygen radicals (a major hallmark of most OXPHOS inhibitors) and decreased the viability of two ovarian cancer cell lines, ID8 and OVCAR5. Finally, data from the pipeline were used to train random forest and support vector classifiers that effectively prioritized OXPHOS inhibitory compounds within a held-out test set (ROCAUC 0.962 and 0.927, respectively) and on another set containing 44 documented OXPHOS inhibitors outside of the training set (ROCAUC 0.900 and 0.823). This prototype pipeline is extensible and could be adapted for focus screening on other phenotypic targets for which sufficient public data are available.Scientific contributionHere, we describe and apply an assay data mining pipeline to compile, process, filter, and mine public bioassay data. We believe the procedure may be more broadly applied to guide compound selection in early-stage hit finding on novel multi-protein mechanistic or phenotypic targets. To demonstrate the utility of our approach, we apply a data mining strategy on a large set of public assay data to find drug-like molecules that inhibit oxidative phosphorylation (OXPHOS) as candidates for ovarian cancer therapies.

Fulvic acid mediated highly efficient heterotrophic nitrification-aerobic denitrification by Paracoccus denitrificans XW11 with reduced C/N ratio.

Reducing the C/N ratio requirements for heterotrophic nitrification-aerobic denitrification (HNAD) is crucial for its practical application; however, it remains underexplored. In this study, a highly efficient HNAD bacterium, Paracoccus denitrificans XW11, was isolated. The HNAD characteristics of XW11 were studied, and the redox mediator fulvic acid (FA) was used to reduce the C/N requirements. Whole-genome sequencing revealed multiple denitrification genes in XW11; however, nitrification genes were not identified, because heterotrophic nitrification-related gene sequences were not included in the database. However, the nitrogen removal related enzyme activity test revealed complete nitrification and denitrification pathways. Reverse transcription PCR showed that the membrane-bound nitrate reductase (NarG), rather than the periplasmic nitrate reductase, was responsible for aerobic denitrification. The conventional nitrite reductase (NirS) also does not mediate nitrite denitrification. When the C/N ratio was 10, the ammonia removal efficiency of the Control was 71.71 % and the addition of FA increased it to 86.12 %. Transcriptomic analysis indicated electron flow from the carbon source to FA without proton transmembrane transport, and the presence of FA constructs another electron transfer system. The redox potential of oxidized FA/reduced FA is 0.3679 V, avoiding competition for electrons from Complex III. Thus, ammonia monooxygenase obtains electrons more easily, thereby promoting nitrification. The enzyme activity test of the nitrification process confirmed this view. In addition, NarG expression increased, and the denitrification process was enhanced. Overall, FA improved HNAD efficiency by facilitating electron transfer to the nitrogen dissimilation process, offering a novel approach to reduce the C/N requirement of HNAD.

Electro-mediated cathodic oxygen drives respiration chain electron transfer of electroactive bacteria to enhance refractory organic biological oxidation.

In electro-mediated biological system (EMBS), biological anode and cathode components were incorporated into an anaerobic bioreactor, providing a small amount of oxygen to the cathode as an electron acceptor. Oxygen diffusion also impacts the anode's anaerobic ecological environment. This study unraveled how oxygen influences the metabolism and electron transport chain during the biological oxidation of refractory organics. Under the influence of electromotive force, the straight-chain model pollutant N,N-dimethylformamide (DMF) showed rapid degradation and better ammonification, with maximum rates reaching 0.53 h-1 and 26.6 %, respectively. Elevated electromotive force promoted the enrichment of functional electroactive bacteria on the anode and enhanced the availability of electron storage sites, thereby facilitating electron transfer at the anode-biofilm interface. Conversely, the anodic micro-aerobic environment disrupted the anaerobic microbial community structure, and the competitive interactions among fermentative bacteria and electroactive bacteria inhibited DMF degradation. Metagenomic analysis confirmed that cathodic oxygen up-regulated the pyruvate metabolism and the tricarboxylic acid (TCA) cycle to generate NADH and synthesize ATP. The electromotive force induced by cathodic oxygen accelerated the electron transfer in respiratory chains of electroactive bacteria, driving the oxidation of NADH and enhancing the degradation of organics. This study improves our understanding of the regulatory mechanisms governing metabolic pathways under the influence of cathodic oxygen. It offers potential for developing more efficient EMBS in industrial wastewater pretreatment, ensuring that oxygen is prevented from diffusing to the anode during micro-aeration at the cathode.

Effects of intermittent exposure to hypobaric hypoxia and cold on skeletal muscle regeneration: mitochondrial dynamics, protein oxidation and turnover.

Muscle injuries and the subsequent regeneration events compromise muscle homeostasis at morphological, functional and molecular levels. Among the molecular alterations, those derived from the mitochondrial function are especially relevant. We analysed the mitochondrial dynamics, the redox balance, the protein oxidation and the main protein repairing mechanisms after 9 days of injury in the rat gastrocnemius muscle. During the recovery rats were exposed to intermittent cold exposure (ICE), intermittent hypobaric hypoxia (IHH), and both simultaneous combined stimuli. Non-injured contralateral legs were also analysed to evaluate the specific effects of the three environmental exposures. Our results showed that ICE enhanced mitochondrial adaptation by improving the electron transport chain efficiency during muscle recovery, decreased the expression of regulatory subunit of proteasome and accumulated oxidised proteins. Exposure to IHH did not show mitochondrial compensation or increased protein turnover mechanisms; however, no accumulation of oxidized proteins was observed. Both ICE and IHH, when applied separately, elicited an increased expression of eNOS, which could have played an important role in accelerating muscle recovery. The combined effect of ICE and IHH led to a complex response that could potentially impede optimal mitochondrial function and enhanced the accumulation of protein oxidation. These findings underscore the nuanced role of environmental stressors in the muscle healing process and their implications for optimizing recovery strategies.

Targeting Asparagine Metabolism in Well-Differentiated/Dedifferentiated Liposarcoma.

BACKGROUND: mTORC1 activity is dependent on the presence of micronutrients, including Asparagine (Asn), to promote anabolic cell signaling in many cancers. We hypothesized that targeting Asn metabolism would inhibit tumor growth by reducing mTORC1 activity in well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS). METHODS: Human tumor metabolomic analysis was utilized to compare abundance of Asn in WD vs. DD LPS. Gene set enrichment analysis (GSEA) compared relative expression among metabolic pathways upregulated in DD vs. WD LPS. Proliferation assays were performed for LPS cell lines and organoid models by using the combination treatment of electron transport chain (ETC) inhibitors with Asn-free media. 13C-Glucose-labeling metabolomics evaluated the effects of combination treatment on nucleotide synthesis. Murine xenograft models were used to assess the effects of ETC inhibition combined with PEGylated L-Asparaginase (PEG-Asnase) on tumor growth and mTORC1 signaling. RESULTS: Asn was enriched in DD LPS compared to WD LPS. GSEA indicated that mTORC1 signaling was upregulated in DD LPS. Within available LPS cell lines and organoid models, the combination of ETC inhibition with Asn-free media resulted in reduced cell proliferation. Combination treatment inhibited nucleotide synthesis and promoted cell cycle arrest. In vivo, the combination of ETC inhibition with PEG-Asnase restricted tumor growth. CONCLUSIONS: Asn enrichment and mTORC1 upregulation are important factors contributing to WD/DD LPS tumor progression. Effective targeting strategies require limiting access to extracellular Asn and inhibition of de novo synthesis mechanisms. The combination of PEG-Asnase with ETC inhibition is an effective therapy to restrict tumor growth in WD/DD LPS.

Mitochondrial bioenergetics of breast cancer.

Breast cancer cells exhibit metabolic heterogeneity based on tumour aggressiveness. Glycolysis and mitochondrial respiration are two major metabolic pathways for ATP production. The oxygen flux, oxygen tension, proton leakage, protonmotive force, inner mitochondrial membrane potential, ECAR and electrochemical proton gradient maintain metabolic homeostasis, ATP production, ROS generation, heat dissipation, and carbon flow and are referred to as "sub-domains" of mitochondrial bioenergetics. Tumour aggressiveness is influenced by these mechanisms, especially when breast cancer cells undergo metastasis. These physiological parameters for healthy mitochondria are as crucial as energy demands for tumour growth and metastasis. The instant energy demands are already elucidated under Warburg effects, while these parameters may have dual functionality to maintain cellular bioenergetics and cellular health. The tumour cell might maintain these mitochondrial parameters for mitochondrial health or avoid apoptosis, while energy production could be a second priority. This review focuses explicitly on the crosstalk between metabolic domains and the utilisation of these parameters by breast cancer cells for their progression. Some major interventions are discussed based on mitochondrial bioenergetics that need further investigation. This review highlights the pathophysiological significance of mitochondrial bioenergetics and the regulation of its sub-domains by breast tumour cells for uncontrolled proliferation.

Multivariate analysis of metabolic state vulnerabilities across diverse cancer contexts reveals synthetically lethal associations.

Targeting the distinct metabolic needs of tumor cells has recently emerged as a promising strategy for cancer therapy. The heterogeneous, context-dependent nature of cancer cell metabolism, however, poses challenges to identifying effective therapeutic interventions. Here, we utilize various unsupervised and supervised multivariate modeling approaches to systematically pinpoint recurrent metabolic states within hundreds of cancer cell lines, elucidate their association with tumor lineage and growth environments, and uncover vulnerabilities linked to their metabolic states across diverse genetic and tissue contexts. We validate key findings via analysis of data from patient-derived tumors and pharmacological screens and by performing genetic and pharmacological experiments. Our analysis uncovers synthetically lethal associations between the tumor metabolic state (e.g., oxidative phosphorylation), driver mutations (e.g., loss of tumor suppressor PTEN), and actionable biological targets (e.g., mitochondrial electron transport chain). Investigating the mechanisms underlying these relationships can inform the development of more precise and context-specific, metabolism-targeted cancer therapies.

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.

MIRO1 controls energy production and proliferation of smooth muscle cells.

Background: The outer mitochondrial Rho GTPase 1, MIRO1, mediates mitochondrial motility within cells, but implications for vascular smooth muscle cell (VSMC) physiology and its roles invascular diseases, such as neointima formation following vascular injury are widely unknown. Methods: An in vivo model of selective Miro1 deletion in VSMCs was generated, and the animals were subjected to carotid artery ligation. The molecular mechanisms relevant to VSMC proliferation were then explored in explanted VSMCs by imaging mitochondrial positioning and cristae structure and assessing the effects on ATP production, metabolic function and interactions with components of the electron transport chain (ETC). Results: MIRO1 was robustly expressed in VSMCs within human atherosclerotic plaques and promoted VSMC proliferation and neointima formation in mice by blocking cell-cycle progression at G1/S, mitochondrial positioning, and PDGF-induced ATP production and respiration; overexpression of a MIRO1 mutant lacking the EF hands that are required for mitochondrial mobility did not fully rescue these effects. At the ultrastructural level, Miro1 deletion distorted the mitochondrial cristae and reduced the formation of super complexes and the activity of ETC complex I. Conclusions: Mitochondrial motility is essential for VSMC proliferation and relies on MIRO1. The EF-hands of MIRO1 regulate the intracellular positioning of mitochondria. Additionally, the absence of MIRO1 leads to distorted mitochondrial cristae and reduced ATP generation. Our findings demonstrate that motility is linked to mitochondrial ATP production. We elucidated two unrecognized mechanisms through which MIRO1 influences cell proliferation by modulating mitochondria: first, by managing mitochondrial placement via Ca2+-dependent EF hands, and second, by affecting cristae structure and ATP synthesis.

Rel-dependent decrease in the expression of ribosomal protein genes by inhibition of the respiratory electron transport chain in Mycobacterium smegmatis.

In this study, we demonstrated that both the expression of most ribosomal protein genes and the amount of ribosomes were decreased in the Δaa 3 mutant of Mycobacterium smegmatis, in which the major terminal oxidase (aa 3 cytochrome c oxidase) of the respiratory electron transport chain (ETC) is inactivated, compared to those in the wild-type strain. Deletion of the rel gene encoding the major (p)ppGpp synthetase in the background of the Δaa 3 mutant restored the reduced expression of ribosomal protein genes, suggesting that inhibition of the respiratory ETC leads to the Rel-dependent stringent response (SR) in this bacterium. Both a decrease in the expression of ribosomal protein genes by overexpression of rel and the increased expression of rel in the Δaa 3 mutant relative to the wild-type strain support the Rel-dependent induction of SR in the Δaa 3 mutant. We also demonstrated that the expression of ribosomal protein genes was decreased in M. smegmatis exposed to respiration-inhibitory conditions, such as KCN and bedaquiline treatment, null mutation of the cytochrome bcc 1 complex, and hypoxia. The MprBA-SigE-SigB regulatory pathway was implicated in both the increased expression of rel and the decreased expression of ribosomal protein genes in the Δaa 3 mutant of M. smegmatis.

The Increase in the Peroxidase Activity of the Cytochrome C with Substitutions in the Universal Binding Site Is Associated with Changes in the Ability to Interact with External Ligands.

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of Cα atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H2O2 and avoid complications of radical formation during the reaction.

Photosynthesis: Genetic Strategies Adopted to Gain Higher Efficiency.

The global challenge of feeding an ever-increasing population to maintain food security requires novel approaches to increase crop yields. Photosynthesis, the fundamental energy and material basis for plant life on Earth, is highly responsive to environmental conditions. Evaluating the operational status of the photosynthetic mechanism provides insights into plants' capacity to adapt to their surroundings. Despite immense effort, photosynthesis still falls short of its theoretical maximum efficiency, indicating significant potential for improvement. In this review, we provide background information on the various genetic aspects of photosynthesis, explain its complexity, and survey relevant genetic engineering approaches employed to improve the efficiency of photosynthesis. We discuss the latest success stories of gene-editing tools like CRISPR-Cas9 and synthetic biology in achieving precise refinements in targeted photosynthesis pathways, such as the Calvin-Benson cycle, electron transport chain, and photorespiration. We also discuss the genetic markers crucial for mitigating the impact of rapidly changing environmental conditions, such as extreme temperatures or drought, on photosynthesis and growth. This review aims to pinpoint optimization opportunities for photosynthesis, discuss recent advancements, and address the challenges in improving this critical process, fostering a globally food-secure future through sustainable food crop production.

Photoelectrochemical Two-Dimensional Electronic Spectroscopy (PEC2DES) of Photosystem I: Charge Separation Dynamics Hidden in a Multichromophoric Landscape.

We present a nonlinear spectroelectrochemical technique to investigate photosynthetic protein complexes. The PEC2DES setup combines photoelectrochemical detection (PEC) that selectively probes the protein photogenerated charges output with two-dimensional electronic spectroscopy (2DES) excitation that spreads the nonlinear optical response of the system in an excitation-detection map. PEC allows us to distinguish the contribution of charge separation (CS) from other de-excitation pathways, whereas 2DES allows us to disentangle congested spectral bands and evaluate the exciton dynamics (decays and coherences) of the photosystem complex. We have developed in operando phase-modulated 2DES by measuring the photoelectrochemical reaction rate in a biohybrid electrode functionalized with a plant photosystem complex I-light harvesting complex I (PSI-LHCI) layer. Optimizing the photoelectrochemical current signal yields reliable linear spectra unequivocally associated with PSI-LHCI. The 2DES signal is validated by nonlinear features like the characteristic vibrational coherence at 750 cm-1. However, no energy transfer dynamics is observed within the 450 fs experimental window. These intriguing results are discussed in the context of incoherent mixing resulting in reduced nonlinear contrast for multichromophoric complexes, such as the 160 chlorophyll PSI. The presented PEC2DES method identifies generated charges unlike purely optical 2DES and opens the way to probe the CS channel in multichromophoric complexes.

Rhodoquinone-dependent electron transport chain is essential for Caenorhabditis elegans survival in hydrogen sulfide environments.

Hydrogen sulfide (H2S) has traditionally been considered an environmental toxin for animal lineages; yet, it plays a signaling role in various processes at low concentrations. Mechanisms controlling H2S in animals, especially in sulfide-rich environments, are not fully understood. The main detoxification pathway involves the conversion of H2S into less harmful forms, through a mitochondrial oxidation pathway. The first step of this pathway oxidizes sulfide and reduces ubiquinone (UQ) through sulfide-quinone oxidoreductase (SQRD/SQOR). Because H2S inhibits cytochrome oxidase and hence UQ regeneration, this pathway becomes compromised at high H2S concentrations. The free-living nematode Caenorhabditis elegans feeds on bacteria and can face high sulfide concentrations in its natural environment. This organism has an alternative ETC that uses rhodoquinone (RQ) as the lipidic electron transporter and fumarate as the final electron acceptor. In this study, we demonstrate that RQ is essential for survival in sulfide. RQ-less animals (kynu-1 and coq-2e KO) cannot survive high H2S concentrations, while UQ-less animals (clk-1 and coq-2a KO) exhibit recovery, even when provided with a UQ-deficient diet. Our findings highlight that sqrd-1 uses both benzoquinones and that RQ-dependent ETC confers a key advantage (RQ regeneration) over UQ in sulfide-rich conditions. C. elegans also faces cyanide, another cytochrome oxidase inhibitor, whose detoxification leads to H2S production, via cysl-2. Our study reveals that RQ delays killing by the HCN-producing bacteria Pseudomonas aeruginosa PAO1. These results underscore the fundamental role that RQ-dependent ETC serves as a biochemical adaptation to H2S environments, and to pathogenic bacteria producing cyanide and H2S toxins.

The Uncoupling Effect of 17ß-Estradiol Underlies the Resilience of Female-Derived Mitochondria to Damage after Experimental TBI.

Current literature finds females have improved outcomes over their male counterparts after severe traumatic brain injury (TBI), while the opposite seems to be true for mild TBI. This begs the question as to what may be driving these sex differences after TBI. Estrogen is thought to be neuroprotective in certain diseases, and its actions have been shown to influence mitochondrial function. Mitochondrial impairment is a major hallmark of TBI, and interestingly, this dysfunction has been shown to be more severe in males than females after brain injury. This suggests estrogen could be playing a role in promoting "mitoprotection" following TBI. Despite the existence of estrogen receptors in mitochondria, few studies have examined the direct role of estrogen on mitochondrial function, and no studies have explored this after TBI. We hypothesized ex vivo treatment of isolated mitochondria with 17ß-estradiol (E2) would improve mitochondrial function after experimental TBI in mice. Total mitochondria from the ipsilateral (injured) and contralateral (control) cortices of male and female mice were isolated 24 h post-controlled severe cortical impact (CCI) and treated with vehicle, 2 nM E2, or 20 nM E2 immediately before measuring reactive oxygen species (ROS) production, bioenergetics, electron transport chain complex (ETC) activities, and ß-oxidation of palmitoyl carnitine. Protein expression of oxidative phosphorylation (OXPHOS) complexes was also measured in these mitochondrial samples to determine whether this influenced functional outcomes with respect to sex or injury. While mitochondrial ROS production was affected by CCI in both sexes, there were other sex-specific patterns of mitochondrial injury 24 h following severe CCI. For instance, mitochondria from males were more susceptible to CCI-induced injury with respect to bioenergetics and ETC complex activities, whereas mitochondria from females showed only Complex II impairment and reduced ß-oxidation after injury. Neither concentration of E2 influenced ETC complex activities themselves, but 20 nM E2 appeared to uncouple mitochondria isolated from the contralateral cortex in both sexes, as well as the injured ipsilateral cortex of females. These studies highlight the significance of measuring mitochondrial dysfunction in both sexes after TBI and also shed light on another potential neuroprotective mechanism in which E2 may attenuate mitochondrial dysfunction after TBI in vivo.

Discovery of antibacterial diketones against gram-positive bacteria.

The rapid rise of antibiotic resistance calls for the discovery of new antibiotics with distinct antibacterial mechanisms. New target mining is indispensable for developing antibiotics. Plant-microbial antibiotics are appealing to underexplored sources due to a dearth of comprehensive understanding of antibacterial activity and the excavation of new targets. Here, a series of phloroglucinol derivatives of plant-root-associated Pseudomonas fluorescens were synthesized for structure-activity relationship analysis. Notably, 2,4-diproylphloroglucinol (DPPG) displayed efficient bactericidal activity against a wide range of gram-positive bacteria. Importantly, mechanistic study exhibits that DPPG binds to type II NADH dehydrogenase (NDH-2), an essential enzyme catalyzing the transfer of electrons from NADH to quinones in the electron transport chain (ETC), blocking electron transfer in S. aureus. Last, we validated the efficacy of DPPG in vivo through animal infection models. Our findings not only provide a distinct antibiotic lead to treat multidrug resistant pathogens but also identify a promising antibacterial target.

SLC30A9: an evolutionarily conserved mitochondrial zinc transporter essential for mammalian early embryonic development.

SLC30A9 (ZnT9) is a mitochondria-resident zinc transporter. Mutations in SLC30A9 have been reported in human patients with a novel cerebro-renal syndrome. Here, we show that ZnT9 is an evolutionarily highly conserved protein, with many regions extremely preserved among evolutionarily distant organisms. In Drosophila melanogaster (the fly), ZnT9 (ZnT49B) knockdown results in acutely impaired movement and drastic mitochondrial deformation. Severe Drosophila ZnT9 (dZnT9) reduction and ZnT9-null mutant flies are pupal lethal. The phenotype of dZnT9 knockdown can be partially rescued by mouse ZnT9 expression or zinc chelator TPEN, indicating the defect of dZnT9 loss is indeed a result of zinc dyshomeostasis. Interestingly, in the mouse, germline loss of Znt9 produces even more extreme phenotypes: the mutant embryos exhibit midgestational lethality with severe development abnormalities. Targeted mutagenesis of Znt9 in the mouse brain leads to serious dwarfism and physical incapacitation, followed by death shortly. Strikingly, the GH/IGF-1 signals are almost non-existent in these tissue-specific knockout mice, consistent with the medical finding in some human patients with severe mitochondrial deficiecny. ZnT9 mutations cause mitochondrial zinc dyshomeostasis, and we demonstrate mechanistically that mitochondrial zinc elevation quickly and potently inhibits the activities of respiration complexes. These results reveal the critical role of ZnT9 and mitochondrial zinc homeostasis in mammalian development. Based on our functional analyses, we finally discussed the possible nature of the so far identified human SLC30A9 mutations.

Extracellular flux assay (Seahorse assay): Diverse applications in metabolic research across biological disciplines.

Metabolic networks are fundamental to cellular processes, driving energy production, biosynthesis, redox regulation, and cellular signaling. Recent advancements in metabolic research tools have provided unprecedented insights into cellular metabolism. Among these tools, the extracellular flux analyzer stands out for its real-time measurement of key metabolic parameters: glycolysis, mitochondrial respiration, and fatty acid oxidation, leading to its widespread use. This review provides a comprehensive summary of the basic principles and workflow of the extracellular flux assay (the Seahorse assay) and its diverse applications. We highlight the assay's versatility across various biological models, including cancer cells, immunocytes, Caenorhabditis elegans, tissues, isolated mitochondria, and three-dimensional structures such as organoids, and summarize key considerations for using extracellular flux assay in these models. Additionally, we discuss the limitations of the Seahorse assay and propose future directions for its development. This review aims to enhance the understanding of extracellular flux assay and its significance in biological studies.

Redox-active vitamin C suppresses human osteosarcoma growth by triggering intracellular ROS-iron-calcium signaling crosstalk and mitochondrial dysfunction.

Pharmacological vitamin C (VC) has gained attention for its pro-oxidant characteristics and selective ability to induce cancer cell death. However, defining its role in cancer has been challenging due to its complex redox properties. In this study, using a human osteosarcoma (OS) model, we show that the redox-active property of VC is critical for inducing non-apoptotic cancer cell death via intracellular reactive oxygen species (ROS)-iron-calcium crosstalk and mitochondrial dysfunction. In both 2D and 3D OS cell culture models, only the oxidizable form of VC demonstrated potent dose-dependent cytotoxicity, while non-oxidizable and oxidized VC derivatives had minimal effects. Live-cell imaging showed that only oxidizable VC caused a surge in cytotoxic ROS, dependent on iron rather than copper. Inhibitors of ferroptosis, a form of iron-dependent cell death, along with classical apoptosis inhibitors, were unable to completely counteract the cytotoxic effects induced by VC. Further pharmacological and genetic inhibition analyses showed that VC triggers calcium release through inositol 1,4,5-trisphosphate receptors (IP3Rs), leading to mitochondrial ROS production and eventual cell death. RNA sequencing revealed down-regulation of genes involved in the mitochondrial electron transport chain and oxidative phosphorylation upon pharmacological VC treatment. Consistently, high-dose VC reduced mitochondrial membrane potential, oxidative phosphorylation, and ATP levels, with ATP reconstitution rescuing VC-induced cytotoxicity. In vivo OS xenograft studies demonstrated reduced tumor growth with high-dose VC administration, concomitant with the altered expression of mitochondrial ATP synthase (MT-ATP). These findings emphasize VC's potential clinical utility in osteosarcoma treatment by inducing mitochondrial metabolic dysfunction through a vicious intracellular ROS-iron-calcium cycle.