MmcA is an electron conduit that facilitates both intracellular and extracellular electron transport in Methanosarcina acetivorans.

Methanogens are a diverse group of Archaea that obligately couple energy conservation to the production of methane. Some methanogens encode alternate pathways for energy conservation, like anaerobic respiration, but the biochemical details of this process are unknown. We show that a multiheme c-type cytochrome called MmcA from Methanosarcina acetivorans is important for intracellular electron transport during methanogenesis and can also reduce extracellular electron acceptors like soluble Fe3+ and anthraquinone-2,6-disulfonate. Consistent with these observations, MmcA displays reversible redox features ranging from -100 to -450 mV versus SHE. Additionally, mutants lacking mmcA have significantly slower Fe3+ reduction rates. The mmcA locus is prevalent in members of the Order Methanosarcinales and is a part of a distinct clade of multiheme cytochromes that are closely related to octaheme tetrathionate reductases. Taken together, MmcA might act as an electron conduit that can potentially support a variety of energy conservation strategies that extend beyond methanogenesis.

Vibrio natriegens as a superior host for the production of c-type cytochromes and difficult-to-express redox proteins.

C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens Vmax X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in Vmax X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, Vmax X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins.

Widespread extracellular electron transfer pathways for charging microbial cytochrome OmcS nanowires via periplasmic cytochromes PpcABCDE.

Extracellular electron transfer (EET) via microbial nanowires drives globally-important environmental processes and biotechnological applications for bioenergy, bioremediation, and bioelectronics. Due to highly-redundant and complex EET pathways, it is unclear how microbes wire electrons rapidly (>106 s-1) from the inner-membrane through outer-surface nanowires directly to an external environment despite a crowded periplasm and slow (<105 s-1) electron diffusion among periplasmic cytochromes. Here, we show that Geobacter sulfurreducens periplasmic cytochromes PpcABCDE inject electrons directly into OmcS nanowires by binding transiently with differing efficiencies, with the least-abundant cytochrome (PpcC) showing the highest efficiency. Remarkably, this defined nanowire-charging pathway is evolutionarily conserved in phylogenetically-diverse bacteria capable of EET. OmcS heme reduction potentials are within 200 mV of each other, with a midpoint 82 mV-higher than reported previously. This could explain efficient EET over micrometres at ultrafast (<200 fs) rates with negligible energy loss. Engineering this minimal nanowire-charging pathway may yield microbial chassis with improved performance.

Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris.

Candida auris has emerged as a problematic fungal pathogen associated with high morbidity and mortality. Amphotericin B (AmB) is the most effective antifungal used to treat invasive fungal candidiasis, with resistance rarely observed among clinical isolates. However, C. auris possesses extraordinary resistant profiles against all available antifungal drugs, including AmB. In our pursuit of potential solutions, we screened a panel of 727 FDA-approved drugs. We identified the proton pump inhibitor lansoprazole (LNP) as a potent enhancer of AmB's activity against C. auris. LNP also potentiates the antifungal activity of AmB against other medically important species of Candida and Cryptococcus. Our investigations into the mechanism of action unveiled that LNP metabolite(s) interact with a crucial target in the mitochondrial respiratory chain (complex III, known as cytochrome bc1). This interaction increases oxidative stress within fungal cells. Our results demonstrated the critical role of an active respiratory function in the antifungal activity of LNP. Most importantly, LNP restored the efficacy of AmB in an immunocompromised mouse model, resulting in a 1.7-log (∼98%) CFU reduction in the burden of C. auris in the kidneys. Our findings strongly advocate for a comprehensive evaluation of LNP as a cytochrome bc1 inhibitor for combating drug-resistant C. auris infections.

Novel Alphaproteobacteria transcribe genes for nitric oxide transformation at high levels in a marine oxygen-deficient zone.

Marine oxygen-deficient zones (ODZs) are portions of the ocean where intense nitrogen loss occurs primarily via denitrification and anammox. Despite many decades of study, the identity of the microbes that catalyze nitrogen loss in ODZs is still being elucidated. Intriguingly, high transcription of genes in the same family as the nitric oxide dismutase (nod) gene from Methylomirabilota has been reported in the anoxic core of ODZs. Here, we show that the most abundantly transcribed nod genes in the Eastern Tropical North Pacific ODZ belong to a new order (UBA11136) of Alphaproteobacteria, rather than Methylomirabilota as previously assumed. Gammaproteobacteria and Planctomycetia also transcribe nod, but at lower relative abundance than UBA11136 in the upper ODZ. The nod-transcribing Alphaproteobacteria likely use formaldehyde and formate as a source of electrons for aerobic respiration, with additional electrons possibly from sulfide oxidation. They also transcribe multiheme cytochrome (here named ptd) genes for a putative porin-cytochrome protein complex of unknown function, potentially involved in extracellular electron transfer. Molecular oxygen for aerobic respiration may originate from nitric oxide dismutation via cryptic oxygen cycling. Our results implicate Alphaproteobacteria order UBA11136 as a significant player in marine nitrogen loss and highlight their potential in one-carbon, nitrogen, and sulfur metabolism in ODZs.IMPORTANCEIn marine oxygen-deficient zones (ODZs), microbes transform bioavailable nitrogen to gaseous nitrogen, with nitric oxide as a key intermediate. The Eastern Tropical North Pacific contains the world's largest ODZ, but the identity of the microbes transforming nitric oxide remains unknown. Here, we show that highly transcribed nitric oxide dismutase (nod) genes belong to Alphaproteobacteria of the novel order UBA11136, which lacks cultivated isolates. These Alphaproteobacteria show evidence for aerobic respiration, using oxygen potentially sourced from nitric oxide dismutase, and possess a novel porin-cytochrome protein complex with unknown function. Gammaproteobacteria and Planctomycetia transcribe nod at lower levels. Our results pinpoint the microbes mediating a key step in marine nitrogen loss and reveal an unexpected predicted metabolism for marine Alphaproteobacteria.

Quorum Sensing Enhances Direct Interspecies Electron Transfer in Anaerobic Methane Production.

Direct interspecies electron transfer (DIET) provides an innovative way to achieve efficient methanogenesis, and this study proposes a new approach to upregulate the DIET pathway by enhancing quorum sensing (QS). Based on long-term reactor performance, QS enhancement achieved more vigorous methanogenesis with 98.7% COD removal efficiency. In the control system, methanogenesis failure occurred at the accumulated acetate of 7420 mg of COD/L and lowered pH of 6.04, and a much lower COD removal of 41.9% was observed. The more significant DIET in QS-enhancing system was supported by higher expression of conductive pili and the c-Cyts cytochrome secretion-related genes, resulting in 12.7- and 10.3-fold improvements. Moreover, QS enhancement also improved the energy production capability, with the increase of F-type and V/A-type ATPase expression by 6.3- and 4.2-fold, and this effect probably provided more energy for nanowires and c-Cyts cytochrome secretion. From the perspective of community structure, QS enhancement increased the abundance of Methanosaeta and Geobacter from 54.3 and 17.6% in the control to 63.0 and 33.8%, respectively. Furthermore, the expression of genes involved in carbon dioxide reduction and alcohol dehydrogenation increased by 0.6- and 7.1-fold, respectively. Taken together, this study indicates the positive effects of QS chemicals to stimulate DIET and advances the understanding of the DIET methanogenesis involved in environments such as anaerobic digesters and sediments.

Colony-Based Electrochemistry Reveals Electron Conduction Mechanisms Mediated by Cytochromes and Flavins in Shewanella oneidensis.

Bacteria utilize electron conduction in their communities to drive their metabolism, which has led to the development of various environmental technologies, such as electrochemical microbial systems and anaerobic digestion. It is challenging to measure the conductivity among bacterial cells when they hardly form stable biofilms on electrodes. This makes it difficult to identify the biomolecules involved in electron conduction. In the present study, we aimed to identify c-type cytochromes involved in electron conduction in Shewanella oneidensis MR-1 and examine the molecular mechanisms. We established a colony-based bioelectronic system that quantifies bacterial electrical conductivity, without the need for biofilm formation on electrodes. This system enabled the quantification of the conductivity of gene deletion mutants that scarcely form biofilms on electrodes, demonstrating that c-type cytochromes, MtrC and OmcA, are involved in electron conduction. Furthermore, the use of colonies of gene deletion mutants demonstrated that flavins participate in electron conduction by binding to OmcA, providing insight into the electron conduction pathways at the molecular level. Furthermore, phenazine-based electron transfer in Pseudomonas aeruginosa PAO1 and flavin-based electron transfer in Bacillus subtilis 3610 were confirmed, indicating that this colony-based system can be used for various bacteria, including weak electricigens.

Pathological and pharmacological functions of the metabolites of polyunsaturated fatty acids mediated by cyclooxygenases, lipoxygenases, and cytochrome P450s in cancers.

Oxylipins have garnered increasing attention because they were consistently shown to play pathological and/or pharmacological roles in the development of multiple cancers. Oxylipins are the metabolites of polyunsaturated fatty acids via both enzymatic and nonenzymatic pathways. The enzymes mediating the metabolism of PUFAs include but not limited to lipoxygenases (LOXs), cyclooxygenases (COXs), and cytochrome P450s (CYPs) pathways, as well as the down-stream enzymes. Here, we systematically summarized the pleiotropic effects of oxylipins in different cancers through pathological and pharmacological aspects, with specific reference to the enzyme-mediated oxylipins. We discussed the specific roles of oxylipins on cancer onset, growth, invasion, and metastasis, as well as the expression changes in the associated metabolic enzymes and the associated underlying mechanisms. In addition, we also discussed the clinical application and potential of oxylipins and related metabolic enzymes as the targets for cancer prevention and treatment. We found the specific function of most oxylipins in cancers, especially the underlying mechanisms and clinic applications, deserves and needs further investigation. We believe that research on oxylipins will provide not only more therapeutic targets for various cancers but also dietary guidance for both cancer patients and healthy humans.

The Interaction Mechanism of Picolinamide Fungicide Targeting on the Cytochrome bc1 Complex and Its Structural Modification.

Picolinamide fungicides, structurally related to UK-2A and antimycin-A, bind into the Qi-site in the bc1 complex. However, the detailed binding mode of picolinamide fungicides remains unknown. In the present study, antimycin-A and UK-2A were selected to study the binding mode of picolinamide inhibitors with four protonation states in the Qi-site by integrating molecular dynamics simulation, molecular docking, and molecular mechanics Generalized Born surface area (MM/GBSA) calculations. Subsequently, a series of new picolinamide derivatives were designed and synthesized to further understand the effects of substituents on the tail phenyl ring. The computational results indicated that the substituted aromatic rings in antimycin-A and UK-2A were the pharmacophore fragments and made the primary contribution when bound to a protein. Compound 9g-hydrolysis formed H-bonds with Hie201 and Ash228 and showed an IC50 value of 6.05 ± 0.24 µM against the porcine bc1 complex. Compound 9c, with a simpler chemical structure, showed higher control effects than florylpicoxamid against cucumber downy mildew and expanded the fungicidal spectrum of picolinamide fungicides. The structural and mechanistic insights obtained from the present study will provide a valuable clue for the future designing of new promising Qi-site inhibitors.

Electron transfer in a crystalline cytochrome with four hemes.

Diffusion of electrons over distances on the order of 100 µm has been observed in crystals of a small tetraheme cytochrome (STC) from Shewanella oneidensis [J. Huang et al. J. Am. Chem. Soc. 142, 10459-10467 (2020)]. Electron transfer between hemes in adjacent subunits of the crystal is slower and more strongly dependent on temperature than had been expected based on semiclassical electron-transfer theory. We here explore explanations for these findings by molecular-dynamics simulations of crystalline and monomeric STC. New procedures are developed for including time-dependent quantum mechanical energy differences in the gap between the energies of the reactant and product states and for evaluating fluctuations of the electronic-interaction matrix element that couples the two hemes. Rate constants for electron transfer are calculated from the time- and temperature-dependent energy gaps, coupling factors, and Franck-Condon-weighted densities of states using an expression with no freely adjustable parameters. Back reactions are considered, as are the effects of various protonation states of the carboxyl groups on the heme side chains. Interactions with water are found to dominate the fluctuations of the energy gap between the reactant and product states. The calculated rate constant for electron transfer from heme IV to heme Ib in a neighboring subunit at 300 K agrees well with the measured value. However, the calculated activation energy of the reaction in the crystal is considerably smaller than observed. We suggest two possible explanations for this discrepancy. The calculated rate constant for transfer from heme I to II within the same subunit of the crystal is about one-third that for monomeric STC in solution.

Extracellular organic disulfide reduction by Shewanella oneidensis MR-1.

Microbial reduction of organic disulfides affects the macromolecular structure and chemical reactivity of natural organic matter. Currently, the enzymatic pathways that mediate disulfide bond reduction in soil and sedimentary organic matter are poorly understood. In this study, we examined the extracellular reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) by Shewanella oneidensis strain MR-1. A transposon mutagenesis screen performed with S. oneidensis resulted in the isolation of a mutant that lost ~90% of its DTNB reduction activity. Genome sequencing of the mutant strain revealed that the transposon was inserted into the dsbD gene, which encodes for an oxidoreductase involved in cytochrome c maturation. Complementation of the mutant strain with the wild-type dsbD partially restored DTNB reduction activity. Because DsbD catalyzes a critical step in the assembly of multi-heme c-type cytochromes, we further investigated the role of extracellular electron transfer cytochromes in organic disulfide reduction. The results indicated that mutants lacking proteins in the Mtr system were severely impaired in their ability to reduce DTNB. These findings provide new insights into extracellular organic disulfide reduction and the enzymatic pathways of organic sulfur redox cycling.IMPORTANCEOrganic sulfur compounds in soils and sediments are held together by disulfide bonds. This study investigates how Shewanella oneidensis breaks apart extracellular organic sulfur compounds. The results show that an enzyme involved in the assembly of c-type cytochromes as well as proteins in the Mtr respiratory pathway is needed for S. oneidensis to transfer electrons from the cell surface to extracellular organic disulfides. These findings have important implications for understanding how organic sulfur decomposes in terrestrial ecosystems.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

Nitrate removal by anammox bacteria utilizing photoexcited electrons via inward extracellular electron transfer channel.

Dissimilatory nitrate reduction to ammonium (DNRA) has been found to occur in some anammox bacteria species, and the DNRA metabolites (nitrite and ammonium) can further be removed to nitrogen from water. However, the activation of DNRA pathway of anammox bacteria is usually limited by the access to electron donors. Herein, we constructed a photosensitized hybrid system combining anammox bacteria (Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia anammoxidans) with CdS nanoparticles semiconductor for energy-efficient NO3- removal. Such photosensitized anammox-CdS hybrid systems achieved NO3- removal with an average efficiency of 88% (the maximum of 91%) and a N2 selectivity of 72%, only with photoexcited electrons as donors. The DNRA-anammox metabolism of anammox bacteria was proved to responsible for NO3- removal via inward extracellular electron transfer channel. The greatly up-regulated genes encoding c-type cytochrome proteins (5 or 11 hemes) in the outer membrane, c-type cytochrome protein (4 hemes) and electron transport protein RnfA-E in the inner membrane, ferredoxin (2Fe-2S) in the cytoplasm and c-type cytochrome bc1 in anammoxosome membrane were supposed to play key roles in the inward extracellular electron transfer pathway. This work provides a novel insight into the design of the biotic-abiotic hybrid photosynthetic systems, and opens a new strategy for light-driven NO3- removal from the perspective of light energy input.

HyperTRCSS: A hyperspectral time-resolved compressive sensing spectrometer for depth-sensitive monitoring of cytochrome-c-oxidase and blood oxygenation.

Significance: Hyperspectral time-resolved (TR) near-infrared spectroscopy offers the potential to monitor cytochrome-c-oxidase (oxCCO) and blood oxygenation in the adult brain with minimal scalp/skull contamination. We introduce a hyperspectral TR spectrometer that uses compressive sensing to minimize acquisition time without compromising spectral range or resolution and demonstrate oxCCO and blood oxygenation monitoring in deep tissue. Aim: Develop a hyperspectral TR compressive sensing spectrometer and use it to monitor oxCCO and blood oxygenation in deep tissue. Approach: Homogeneous tissue-mimicking phantom experiments were conducted to confirm the spectrometer's sensitivity to oxCCO and blood oxygenation. Two-layer phantoms were used to evaluate the spectrometer's sensitivity to oxCCO and blood oxygenation in the bottom layer through a 10 mm thick static top layer. Results: The spectrometer was sensitive to oxCCO and blood oxygenation changes in the bottom layer of the two-layer phantoms, as confirmed by concomitant measurements acquired directly from the bottom layer. Measures of oxCCO and blood oxygenation by the spectrometer were highly correlated with "gold standard" measures in the homogeneous and two-layer phantom experiments. Conclusions: The results show that the hyperspectral TR compressive sensing spectrometer is sensitive to changes in oxCCO and blood oxygenation in deep tissue through a thick static top layer.

[Add-on value of plasma assays during prescription of antipsychotics]. / Intérêt des dosages plasmatiques lors de la prescription des antipsychotiques.

The plasma dosage of antipsychotics is justified in many clinical situations, such as the existence of surprising side effects depending on the dose of administered antipsychotic or, in the event of a lack of therapeutic efficacy. This method allows the clinician to get out of the pure empiricism, trial and error, of his/her supposed beliefs concerning patient compliance and, above all, to base his/her decisions on objective elements in the psychopharmacological field. However, it is clear that this inexpensive and easily accessible technology is not used very often. In the event of a result of the plasma dosage higher or lower than the normal values, it will first be necessary to exclude a phenomenon of drug interactions. Then, the calculation of the metabolic ratio will make it possible to suspect the existence of a particular genetic polymorphism of metabolism (poor metabolizer? ultra-rapid metabolizer?) of the concerned cytochrome, which can then be confirmed by genotyping oriented towards this cytochrome.

Le dosage plasmatique des antipsychotiques est justifié dans de nombreuses situations cliniques, comme l'existence d'effets secondaires surprenants en fonction de la dose d'antipsychotique administrée, ou encore, en cas de manque d'efficacité thérapeutique. Cette méthode permet au clinicien de sortir de l'empirisme pur, essai-erreur, de ses supposées croyances concernant l'observance du patient et, surtout, de baser ses décisions sur les éléments objectifs dans le domaine psychopharmacologique. Pourtant, force est de constater que cette technologie peu coûteuse et facilement accessible n'est que trop peu souvent utilisée. En cas de résultat du dosage plasmatique supérieur ou inférieur à la norme, il conviendra, tout d'abord, d'exclure un phénomène d'interactions médicamenteuses. Ensuite, le calcul du ratio métabolique permettra de soupçonner éventuellement l'existence d'un polymorphisme génétique de métabolisation particulier (métaboliseur lent ? métaboliseur ultra-rapide ?) du cytochrome concerné, qui pourra alors être confirmé par un génotypage orienté vers ce cytochrome.

Bidirectional extracellular electron transfer pathways of Geobacter sulfurreducens biofilms: Molecular insights into extracellular polymeric substances.

The basis for bioelectrochemical technology is the capability of electroactive bacteria (EAB) to perform bidirectional extracellular electron transfer (EET) with electrodes, i.e. outward- and inward-EET. Extracellular polymeric substances (EPS) surrounding EAB are the necessary media for EET, but the biochemical and molecular analysis of EPS of Geobacter biofilms on electrode surface is largely lacked. This study constructed Geobacter sulfurreducens-biofilms performing bidirectional EET to explore the bidirectional EET mechanisms through EPS characterization using electrochemical, spectroscopic fingerprinting and proteomic techniques. Results showed that the inward-EET required extracellular redox proteins with lower formal potentials relative to outward-EET. Comparing to the EPS extracted from anodic biofilm (A-EPS), the EPS extracted from cathodic biofilm (C-EPS) exhibited a lower redox activity, mainly due to a decrease of protein/polysaccharide ratio and α-helix content of proteins. Furthermore, less cytochromes and more tyrosine- and tryptophan-protein like substances were detected in C-EPS than in A-EPS, indicating a diminished role of cytochromes and a possible role of other redox proteins in inward-EET. Proteomic analysis identified a variety of redox proteins including cytochrome, iron-sulfur clusters-containing protein, flavoprotein and hydrogenase in EPS, which might serve as an extracellular redox network for bidirectional EET. Those redox proteins that were significantly stimulated in A-EPS and C-EPS might be essential for outward- and inward-EET and warranted further research. This work sheds light on the mechanism of bidirectional EET of G. sulfurreducens biofilms and has implications in improving the performance of bioelectrochemical technology.

Genome wide and comprehensive analysis of the cytochrome P450 (CYPs) gene family in Pyrus bretschneideri: Expression patterns during Sporidiobolus pararoseus Y16 enhanced with ascorbic acid (VC) treatment.

Cytochrome P450s (CYPs) constitute the largest group of enzymes in plants and are involved in a variety of processes related to growth and protection. However, the CYP gene superfamily in pear (Pyrus bretschneideri) and their characteristics is unclear. Through a comprehensive genome-wide analysis, this article identified a total of 74 CYP genes in the P. bretschneideri genome, which were categorized into fourteen families. Motif analysis reveals that most of the ten motifs predicted were with the p450 conserved domain. The majority of the CYP genes have exon arrangements. Furthermore, promoter analysis unveiled a multitude of cis-acting elements associated with diverse responsiveness including hormones, light responsive, anoxic specific inducibility and anaerobic induction. Analysis of the transcriptome data reveal that about 80% of the pear CYPs genes were upregulated and they were positively correlated with the antioxidant's parameters such as total flavonoids and total phenol content as well as ABTS and DPPH radicals. RT-qPCR analysis confirmed that the CYP genes could be regulated in pear. Collectively, our results reveal comprehensive insights into the CYP superfamily in pear and make a valuable contribution to the ongoing process of functional validation.

Dapagliflozin-entresto protected kidney from renal hypertension via downregulating cell-stress signaling and upregulating SIRT1/PGC-1α/Mfn2-medicated mitochondrial homeostasis.

This study tested whether combined dapagliflozin and entresto would be superior to mere one therapy on protecting the residual renal function and integrity of kidney parenchyma in hypertensive kidney disease (HKD) rat. In vitro results showed that the protein expressions of oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/oxidized-protein/cytosolic-cytochrome-C)/apoptotic (mitochondrial-Bax/cleaved caspeases 3, 9)/cell-stress (p-ERK/p-JNK/p-p38) biomarkers were significantly increased in H2O2-treated NRK-52E cells than those of controls that were reversed by dapagliflozin or entresto treatment. Adult-male SD rats (n = 50) were equally categorized into group 1 (sham-operated-control), group 2 (HKD by 5/6 nephrectomy + DOCA-salt/25 mg/kg/subcutaneous injection/twice weekly), group 3 (HKD + dapagliflozin/orally, 20 mg/kg/day for 4 weeks since day 7 after HKD induction), group 4 (HKD + entresto/orally, 100 mg/kg/day for 4 weeks since day 7 after HKD induction), and group 5 (HKD + dapagliflozin + entresto/the procedure and treatment strategy were identical to groups 2/3/4). By day 35, circulatory levels of blood-urine-nitrogen (BUN)/creatinine and urine protein/creatinine ratio were lowest in group 1, highest in group 2, and significantly lower in group 5 than in groups 3/4, but no difference between groups 3/4. Histopathological findings showed the kidney injury score/fibrotic area/cellular expressions of oxidative-stress/kidney-injury-molecule (8-OHdG+/KIM-1+) exhibited an identical trend, whereas the cellular expressions of podocyte components (synaptopodin/ZO-1/E-cadherin) exhibited an opposite pattern of BUN level among the groups. The protein expressions of oxidative stress/mitochondrial-damaged (NOX-1/NOX-2/oxidized protein/cytosolic-cytochrome-C/cyclophilin-D)/apoptotic (mitochondrial-Bax/cleaved-caspase 3)/mitochondrial-fission (PINK1/Parkin/p-DRP1)/autophagic (LC3BII/LC3BI ratio, Atg5/beclin-1)/MAPK-family (p-ERK/p-JNK/p-p38) biomarkers displayed a similar pattern, whereas the protein expression of mitochondria-biogenesis signaling (SIRT1/PGC-1α-Mfn2/complex I-V) displayed an opposite pattern of BUN among the groups. In conclusion, combined dapagliflozin-entresto therapy offered additional benefits on protecting the residual kidney function and architectural integrity in HKD rat.

[Cytochrome bd as Antioxidant Redox Enzyme].

One of the main functions of enzyme complexes that constitute electron transport (respiratory) chains of organisms is to maintain cellular redox homeostasis by oxidizing reducing equivalents, NADH and quinol. Cytochrome bd is a unique terminal oxidase of the chains of many bacteria including pathogenic species. This redox enzyme couples the oxidation of ubiquinol or menaquinol by molecular oxygen to the generation of proton motive force, a universal energy currency. The latter is used by the organism to produce ATP, another cellular energy currency, via oxidative phosphorylation. Escherichia coli contains two bd-type oxidases, bd-I and bd-II, encoded by the cydAB and appCB operons, respectively. Surprisingly, both bd enzymes make a further contribution to molecular mechanisms of maintaining the appropriate redox balance in the bacterial cell by means of elimination of reactive oxygen species, such as hydrogen peroxide. This review summarizes recent data on the redox-modulated H2O2-scavenging activities of cytochromes bd-I and bd-II from E. coli. The possibility of such antioxidant properties in cytochromes bd from other bacteria is also discussed.

Repurposing of Tuberculosis Drug Candidates for the Treatment of Mycobacterium ulcerans Disease.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M. ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M. ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M. tuberculosis, classical lineage M. ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M. ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.