Efflux pumps mediate changes to fundamental bacterial physiology via membrane potential.

Efflux pumps are well known to be an important mechanism for removing noxious substances such as antibiotics from bacteria. Given that many antibiotics function by accumulating inside bacteria, efflux pumps contribute to resistance. Efflux pump inactivation is a potential strategy to combat antimicrobial resistance, as bacteria would not be able to pump out antibiotics. We recently discovered that the impact of loss of efflux function is only apparent in actively growing cells. We demonstrated that the global transcriptome of Salmonella Typhimurium is drastically altered during slower growth leading to stationary-phase cells having a remodeled, less permeable envelope that prevents antibiotics entering the cell. Here, we investigated the effects of deleting the major efflux pump of Salmonella Typhimurium, AcrB, on global gene transcription across growth. We revealed that an acrB knockout entered stationary phase later than the wild-type strain SL1344 and displayed increased and prolonged expression of genes responsible for anaerobic energy metabolism. We devised a model linking efflux and membrane potential, whereby deactivation of AcrB prevents influx of protons across the inner membrane and thereby hyperpolarization. Knockout or deactivation of AcrB was demonstrated to increase membrane potential. We propose that the global transcription regulator ArcBA senses changes to the redox state of the quinol pool (linked to the membrane potential of the bacterium) and coordinates the shift from exponential to stationary phase via the key master regulators RpoS, Rsd, and Rmf. Inactivation of efflux pumps therefore influences the fundamental physiology of Salmonella, with likely impacts on multiple phenotypes.IMPORTANCEWe demonstrate for the first time that deactivation of efflux pumps brings about changes to gross bacterial physiology and metabolism. Rather than simply being a response to noxious substances, efflux pumps appear to play a key role in maintenance of membrane potential and thereby energy metabolism. This discovery suggests that efflux pump inhibition or inactivation might have unforeseen positive consequences on antibiotic activity. Given that stationary-phase bacteria are more resistant to antibiotic uptake, late entry into stationary phase would prolong antibiotic accumulation by bacteria. Furthermore, membrane hyperpolarization could result in increased generation of reactive species proposed to be important for the activity of some antibiotics. Finally, changes in gross physiology could also explain the decreased virulence of efflux mutants.

Modeling riboflavin transporter deficiency type 2: from iPSC-derived motoneurons to iPSC-derived astrocytes.

Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes.

Passively administered fluoxetine reaches the juvenile brain of FSL rats and reduces antioxidant defences, without altering serotonin turnover.

BACKGROUND: Fluoxetine is present in breast milk, yet it is unclear to what extent it, or its active metabolite, norfluoxetine, reaches the brain of the infant and what the effects of such exposure on neurobiological processes are. We therefore aimed to quantify the concentration of passively administered fluoxetine and norfluoxetine in the whole brains of exposed Flinders sensitive line (FSL) offspring and establish their influence on serotonergic function and redox status. METHODS: Adult FSL dams received fluoxetine (10 mg/kg/day), or placebo for fourteen days, beginning on postpartum day 04. Offspring were passively exposed to fluoxetine until postnatal day 18 and euthanized on postnatal day 22. Whole brain fluoxetine, norfluoxetine, serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and reduced (GSH) and oxidized glutathione (GSSG) concentrations were measured via liquid chromatography-mass spectrometry (LC-MS) analysis. RESULTS: Whole-brain serotonin and 5-hydroxyindoleacetic acid concentrations, and serotonin turnover (5-HIAA/5-HT) were comparable between strains. Treatment-naïve FSL rats had lower GSH and higher GSSG whole-brain concentrations, relative to FRL controls, and an overall decreased GSH/GSSG ratio. Passively administered fluoxetine resulted in undetectable whole-brain concentrations, while norfluoxetine averaged 41.28 ± 6.47 ng/g. Serotonin turnover of FSL rats was unaffected by passively administered fluoxetine, while redox status (GSH/GSSG) was decreased. CONCLUSION: Our findings confirm that passively administered fluoxetine reaches the infant brain in the form of norfluoxetine and may manipulate processes of oxidative stress regulation. Further studies into the long-term bio-behavioural effects are however needed to effectively inform breast feeding mothers on the safety of antidepressant-use.

Transmembrane Graphene as an Electron Tunnel to Regulate the Intracellular Redox State.

Cellular redox homeostasis is essential for maintaining cellular activities, such as DNA synthesis and gene expression. Inspired by this, new therapeutic interventions have been rapidly developed to modulate the intracellular redox state using artificial transmembrane electron transport. However, current approaches that rely on external electric field polarization can disrupt cellular functions, limiting their in vivo application. Therefore, it is crucial to develop novel electric-field-free modulation methods. In this work, we for the first time found that graphene could spontaneously insert into living cell membranes and serve as an electron tunnel to regulate intracellular reactive oxygen species and NADH based on the spontaneous bipolar electrochemical reaction mechanism. This work provides a wireless and electric-field-free approach to regulating cellular redox states directly and offers possibilities for biological applications such as cell process intervention and treatment for neurodegenerative diseases.

Alginate oligosaccharides exert protective effects on hydrogen peroxide-induced senescence in H9C2 cardiomyocytes by regulating the redox state of cells.

Aging is a known independent risk factor for several cardiovascular diseases. Here, we evaluated potential effects and possible mechanisms through which alginate oligosaccharides (AOS) affect hydrogen peroxide (H2O2)-induced senescence in H9C2 cardiomyocytes. A series of AOS molecules, including oligoM, oligoG, M-5, and G-5, were investigated. AOS significantly decreased SA-ß-gal and DAPI-stained positive cells, downregulated p53 and p21 (aging-related markers) expression, and eventually protected H9C2 cells from H2O2-induced senescence. AOS decreased reactive oxygen species and malondialdehyde production, recovered mitochondrial function, and alleviated the oxidative stress state by regulating PGC-1α and NADPH oxidase subunit expression. Furthermore, AOS treatment restored the expression of antioxidant enzymes in senescent H9C2 cells. Thus, our results show in vitro evidence that AOS alleviate senescence in H9C2 cells by regulating the redox state; thus, AOS may be an effective therapeutic agent that could protect against cardiomyocyte senescence.

Effects of polyvinyl chloride and low-density polyethylene microplastics on oxidative stress and mitochondria function of earthworm (Eisenia fetida).

Plastics are widely used worldwide due to their convenience. However, microplastics (MPs) accumulation poses a serious threat to ecosystem health. Therefore, understanding the effects of MPs on living organisms within their native ecosystem is crucial. Previous studies have primarily focused on the impacts of MPs in aquatic environments, whereas the effects of MPs on terrestrial ecosystems have remained largely understudied. Therefore, our study assessed the impacts of MPs on soil ecosystems by characterizing their toxic effects on earthworms (Eisenia fetida). Here, we exposed earthworms to two representative plastics within soil environments: polyvinyl chloride (PVC) and low-density polyethylene (LDPE). Given the known link between MPs and oxidative stress, we next quantified oxidative stress markers and mitochondrial function to assess the effects of MPs on the redox metabolism of earthworms. Mitochondria are crucial metabolic organelles that generate reactive oxygen species via uncontrolled ATP production. Our findings demonstrated that MPs exert different effects depending on their type. Neither the PVC-exposed groups nor the LDPE-exposed groups exhibited changes in oxidative stress, as worked by the action of superoxide dismutase (SOD) and glutathione (GSH). While treatment of the two types of MP did not significantly affect the amount of reactive oxygen species/reactive nitrogen species (ROS/RNS) generated, PVC exhibited a more pronounced effect on antioxidant system compared to LDPE. However, mitochondrial function was markedly decreased in the group exposed to high LDPE concentrations, suggesting that the examined LDPE concentrations were too low to activate compensatory mechanisms. Collectively, our findings demonstrated that exposure of MPs not only influences the antioxidant defense mechanisms of earthworms but also alters their mitochondrial function depending on their types.

Navigating the redox landscape: reactive oxygen species in regulation of cell cycle.

Objectives: To advance our knowledge of disease mechanisms and therapeutic options, understanding cell cycle regulation is critical. Recent research has highlighted the importance of reactive oxygen species (ROS) in cell cycle regulation. Although excessive ROS levels can lead to age-related pathologies, ROS also play an essential role in normal cellular functions. Many cell cycle regulatory proteins are affected by their redox status, but the precise mechanisms and conditions under which ROS promote or inhibit cell proliferation are not fully understood.Methods: This review presents data from the scientific literature and publicly available databases on changes in redox state during the cell cycle and their effects on key regulatory proteins.Results: We identified redox-sensitive targets within the cell cycle machinery and analysed different effects of ROS (type, concentration, duration of exposure) on cell cycle phases. For example, moderate levels of ROS can promote cell proliferation by activating signalling pathways involved in cell cycle progression, whereas excessive ROS levels can induce DNA damage and trigger cell cycle arrest or cell death.Discussion: Our findings encourage future research focused on identifying redox-sensitive targets in the cell cycle machinery, potentially leading to new treatments for diseases with dysregulated cell proliferation.

Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control.

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust, soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, incorporates lysis enzymes for purification, and can also integrate dynamic expression of protein folding catalysts. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

Docosahexaenoic Acid Coordinating with Sodium Selenite Promotes Paraptosis in Colorectal Cancer Cells by Disrupting the Redox Homeostasis and Activating the MAPK Pathway.

Tumor cells are characterized by a delicate balance between elevated oxidative stress and enhanced antioxidant capacity. This intricate equilibrium, maintained within a threshold known as redox homeostasis, offers a unique perspective for cancer treatment by modulating reactive oxygen species (ROS) levels beyond cellular tolerability, thereby disrupting this balance. However, currently used chemotherapy drugs require larger doses to increase ROS levels beyond the redox homeostasis threshold, which may cause serious side effects. How to disrupt redox homeostasis in cancer cells more effectively remains a challenge. In this study, we found that sodium selenite and docosahexaenoic acid (DHA), a polyunsaturated fatty acid extracted from marine fish, synergistically induced cytotoxic effects in colorectal cancer (CRC) cells. Physiological doses of DHA simultaneously upregulated oxidation and antioxidant levels within the threshold range without affecting cell viability. However, it rendered the cells more susceptible to reaching the upper limit of the threshold of redox homeostasis, facilitating the elevation of ROS levels beyond the threshold by combining with low doses of sodium selenite, thereby disrupting redox homeostasis and inducing MAPK-mediated paraptosis. This study highlights the synergistic anticancer effects of sodium selenite and DHA, which induce paraptosis by disrupting redox homeostasis in tumor cells. These findings offer a novel strategy for more targeted and less toxic cancer therapies for colorectal cancer treatment.

Mitochondrial Dihydrolipoamide Dehydrogenase (mtLPD1): Expression, Purification, Activity, and Redox Regulation.

Mitochondrial dihydrolipoamide dehydrogenase (mtLPD1) is a central enzyme in primary carbon metabolism, since its function is required to drive four multienzymes involved in photorespiration, the tricarboxylic acid (TCA) cycle, and the degradation of branched-chain amino acids. However, in illuminated, photosynthesizing tissue a vast amount of mtLPD1 is necessary for glycine decarboxylase (GDC), the key enzyme of photorespiration. In light of the shared role, the functional characterization of mtLPD1 is necessary to understand how the three pathways might interact under different environmental scenarios. This includes the determination of the biochemical properties and all potential regulatory mechanisms, respectively. With regards to the latter, regulation can occur through multiple levels including effector molecules, cofactor availability, or posttranslational modifications (PTM), which in turn decrease or increase the activity of each enzymatic reaction. Gaining a comprehensive overview on all these aspects would ultimately facilitate the interpretation of the metabolic interplay of the pathways within the whole subcellular network or even function as a proof of concept for genetic engineering approaches. Here, we describe the typical workflow how to clone, express, and purify plant mtLPD1 for biochemical characterization and how to analyze potential redox regulatory mechanisms in vitro and in planta.

Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry.

Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.

PM2.5 toxicity in blood impairs cardiac redox balance and promotes mitochondrial dysfunction in rat heart that further aggravates ischemia reperfusion injury by modulating PI3K/AKT/mTOR/NF-kB signaling axis.

According to the pathophysiological mechanisms linking particulate matter (PM2.5) exposure and cardiovascular diseases, PM2.5 may directly translocate into the blood stream and remote target organs and thereby induce cardiovascular effects. The toxicity of PM2.5 is known to induce oxidative stress in pulmonary tissue, but its impact on the redox state in heart (distant organ) is unknown and how it modulates the cardiac response to ischemia reperfusion (IR) remains unclear. In the present study, we evaluated the toxic effect of PM2.5 on cardiac physiology in the presence and absence of IR after introducing PM2.5 into the blood. Female Wistar rats were injected with diesel particulate matter (DPM) via i.p & i.v routes at a concentration of 10 µg/ml. The toxic impact of PM2.5 not only adversely affects the cardiac ultra-structure (leading to nuclear infiltration, edema, irregularities in heart muscle and nuclear infiltration), but also altered the cellular redox balance, elevated inflammation and promoted the upregulation of proapoptotic mediator genes at the basal level of myocardium. The results showed alterations in cardiac ultrastructure, elevated oxidative stress and significant redox imbalance, increased inflammation and proapoptotic mediators at the basal level of myocardium. Moreover, the cardioprotective pro survival signaling axis was declined along with an increased NF-kB activation at the basal level. IR inflicted further injury with deterioration of cardiac hemodynamic indices (Heart rate [HR], Left ventricular developed pressure [LVDP], Left ventricular end-diastolic pressure [LVEDP] and rate pressure product [RPP]) along with prominent inactivation of signaling pathways. Furthermore, the levels of GSH/GSSG, NADH/NAD, NADPH/NADP were significantly low along with increased lipid peroxidation in mitochondria of PM2.5 treated IR rat hearts. This observation was supported by downregulation of glutaredoxin and peroxiredoxin genes in the myocardium. Similarly the presence of oxidative stress inducing metals was found at a higher concentration in cardiac mitochondria. Thus, the toxic impact of PM2.5 in heart augment the IR associated pathological changes by altering the physiological response, initiating cellular metabolic alterations in mitochondria and modifying the signaling molecules.

Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity.

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd-EtfAB (BCD) complex was overexpressed, the resulting strain PRH-B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45-0.51 g/L/h and 0.46-0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin-NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

Use of Optical Redox Imaging to Quantify Alveolar Macrophage Redox State in Infants: Proof of Concept Experiments in a Murine Model and Human Tracheal Aspirates Samples.

Emerging data indicate that lung macrophages (LM) may provide a novel biomarker to classify disease endotypes in bronchopulmonary dysplasia (BPD), a form of infant chronic lung disease, and that augmentation of the LM phenotype may be a potential therapeutic target. To contribute to this area of research, we first used Optical Redox Imaging (ORI) to characterize the responses to H2O2-induced oxidative stress and caffeine treatment in an in vitro model of mouse alveolar macrophages (AM). H2O2 caused a dose-dependent decrease in NADH and an increase in FAD-containing flavoproteins (Fp) and the redox ratio Fp/(NADH + Fp). Caffeine treatment did not affect Fp but significantly decreased NADH with doses of ≥50 µM, and 1000 µM caffeine treatment significantly increased the redox ratio and decreased the baseline level of mitochondrial ROS (reactive oxygen species). However, regardless of whether AM were pretreated with caffeine or not, the mitochondrial ROS levels increased to similar levels after H2O2 challenge. We then investigated the feasibility of utilizing ORI to examine macrophage redox status in tracheal aspirate (TA) samples obtained from premature infants receiving invasive ventilation. We observed significant heterogeneity in NADH, Fp, Fp/(NADH + Fp), and mitochondrial ROS of the TA macrophages. We found a possible positive correlation between gestational age and NADH and a negative correlation between mean airway pressure and NADH that provides hypotheses for future testing. Our study demonstrates that ORI is a feasible technique to characterize macrophage redox state in infant TA samples and supports further use of this method to investigate lung macrophage-mediated disease endotypes in BPD.

Efficacy of Medical Ozone for Treatment of Chronic Musculoskeletal Pain with Abnormal Mitochondrial Redox State: Prospective Randomized Clinical Trial.

BACKGROUND: Chronic primary musculoskeletal pain is multifaceted and 20% of the adult population lives with severe chronic pain and experience symptoms such as intense pain, depression, weakness, sleep problems, decreased quality of life and decreased emotional well-being. OBJECTIVES: This paper studies the efficacy of trigger point injections with ozone compared to standard steroid injection or combination therapy for the treatment of chronic musculoskeletal pain in patients with abnormal mitochondrial redox state. STUDY DESIGN: This is a prospective randomized clinical study conducted with 51 patients experiencing chronic musculoskeletal pain. SETTING: Medical Research Institute Hospital, Alexandria University. METHODS: By computer-generated random numbers the 51 patients were divided into 3 groups. Group A (17 patients) received ozone injection, group B (17 patients) received betamethasone injection and group C (17 patients) received combined ozone and betamethasone injections. The groups were compared based on the intensity of pain and correction of mitochondrial redox state of the patients. RESULTS: Three days after intervention, the visual analog scale (VAS) scores reported by patients were lower in group A compared to group B (with a mean difference 1.27, 95% confidence interval (CI) of 0.15-2.39 (P < 0.02). One and 3 weeks after intervention, VAS scores of patients were lower in groups A and C compared to group B. At one week the mean difference between A and B was 1.2, with a 95% CI of 0.15-2.25 (P < 0.02) and the mean difference between C and B was 1.73 with a 95% CI of 0.69-2.78 (P < 0.001). At 3 weeks the mean difference between A and B was 1.5 with a 95% CI of 0.2-2.87 (P < 0.01) and the mean difference between C and B was 2.27 with a 95% CI of 0.93-3.60 (P < 0.0001). The reduced/oxidized glutathione ratio after intervention was higher in groups A and C compared to group B (P > 0.008). The mitochondrial copy number was higher in group A compared to group B (P < 0.002). LIMITATION: This study didn't allow for the comparison of the experimental groups with a placebo or control group for musculoskeletal pain conditions in orderto establish the role of an abnormal mitochondrial redox state on the pathogenesis of patients from an ethical view. CONCLUSIONS: Ozone therapy or combined ozone and betamethasone treatment are  effective techniques for management of pain since it produced a significant reduction of muscle pain and increase of the pain free interval experienced by patients. Ozone therapy causes pain improvement which increases with time and it improves muscle oxygenation and mitochondrial function. TRIAL REGISTRATION: This study was approved by the Ethics Committee of Medical Research Institute (IORH: IOR 00088812) and was registered at the Pan African Clinical Trial Registry (www.pactr.org) under the identification number PACTR201908620943471. The registration this experiment started on 07/08/2019. This study's protocol followed the CONSORT guidelines and was performed under the relevant guidelines.

The tissue-specific nature of physiological zebrafish mitochondrial bioenergetics.

Zebrafish are a powerful tool to study a myriad of experimental conditions, including mitochondrial bioenergetics. Considering that mitochondria are different in many aspects depending on the tissue evaluated, in the zebrafish model there is still a lack of this investigation. Especially for juvenile zebrafish. In the present study, we examined whether different tissues from zebrafish juveniles show mitochondrial density- and tissue-specificity comparing brain, liver, heart, and skeletal muscle (SM). The liver and brain complex IV showed the highest O2 consumption of all ETC in all tissues (10x when compared to other respiratory complexes). The liver showed a higher potential for ROS generation. In this way, the brain and liver showed more susceptibility to O2- generation when compared to other tissues. Regarding Ca2+ transport, the brain showed greater capacity for Ca2+ uptake and the liver presented low Ca2+ uptake capacity. The liver and brain were more susceptible to producing NO. The enzymes SOD and Catalase showed high activity in the brain, whereas GPx showed higher activity in the liver and CS in the SM. TEM reveals, as expected, a physiological diverse mitochondrial morphology. The essential differences between zebrafish tissues investigated probably reflect how the mitochondria play a diverse role in systemic homeostasis. This feature may not be limited to normal metabolic functions but also to stress conditions. In summary, mitochondrial bioenergetics in zebrafish juvenile permeabilized tissues showed a tissue-specificity and a useful tool to investigate conditions of redox system imbalance, mainly in the liver and brain.

Quantitative Optical Redox Imaging of Melanoma Xenografts with Different Metastatic Potentials.

To develop imaging biomarkers for tumors aggressiveness, our previous optical redox imaging (ORI) studies of the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp, containing flavin adenine dinucleotide, i.e., FAD) in tumor xenografts of human melanoma associated the high optical redox ratio (ORR = Fp/(Fp + NADH)) and its heterogeneity to the high invasive/metastatic potential, without having reported quantitative results for NADH and Fp. Here, we implemented a calibration procedure to facilitate imaging the nominal concentrations of tissue NADH and Fp in the mouse xenografts of two human melanoma lines, an indolent less metastatic A375P and a more metastatic C8161. Images of the redox indices (NADH, Fp, ORR) revealed the existence of more oxidized areas (OAs) and more reduced areas (RAs) within individual tumors. ORR was found to be higher and NADH lower in C8161 compared to that of A375P xenografts, both globally for the whole tumors and locally in OAs. The ORR in the OA can differentiate xenografts with a higher statistical significance than the global averaged ORR. H&E staining of the tumors indicated that the redox differences we identified were more likely due to intrinsically different cell metabolism, rather than variations in cell density.

Clinicopathological studies on ulcerative lymphangitis in cattle: Alterations in serum inflammatory cytokines, anti-microbial, organs functions, and oxidative stress-related biomarkers.

Background: Affection with Corynebacterium pseudotuberculosis (C. pseudotuberculosis) and development of cellulitis and/or abscess formation with cutaneous lymphangitis in cattle is rare to some extent, so literature about the biochemical changes that would accompany this infection is rare. Aim: In this context, the present study was designed to screen the effect of the infection with C. pseudotuberculosis cutaneous lymphangitis on the release of some immune molecules, organ functions, and redox state in Baladi cows. Methods: Fourteen Baladi cows from a small dairy farm in El-Behira, Egypt, were selected to complete this study. After bacteriological culture confirmation, seven of them were found suffering from cutaneous lesions due to infection with C. pseudotuberculosis (Diseased group), while the others were healthy (Healthy group). Serum samples were obtained to evaluate the presumptive changes in some clinicopathological parameters. Results: Serum analysis revealed a significant decrease in the levels of interferon-gamma and interleukin-17 as well as a significant decrement in the concentration of beta-defensin (ß-defensin) and lipocalin-2. While serum level of interleukin-10 recorded a significant increase in these animals when compared to healthy control animals. Concurrently, the affected animals recorded a significant elevation in serum levels of hepato-cardiac enzymes, urea, and creatinine in addition to disturbance in the serum redox state. Conclusion: In conclusion, infection with C. pseudotuberculosis cattle may disturb the defensive immune state, body organ function, and redox state of the animals.

Shining Light into a "Black Box": Essential Rationale Underlying Multiphase Flash Methodology.

The world we live in is very fragile. Sustainable food production is increasingly under intense pressure due to changing environmental conditions on many levels. Understanding the complexities of how to optimize food production under increasingly deleterious environmental conditions is dependent upon accurate and detailed analyses of plant productivity from the molecular-to-the-remote scales. One method that can link many of these scales has been around for decades, namely, pulse amplitude modulation (PAM) chlorophyll a fluorescence. This technique is used to measure an assortment of important parameters based on chlorophyll a fluorescence. One of the parameters measured by this method is termed the steady state maximum fluorescence yield ( Φ Fm ' ). This parameter, while extremely informative when used to quantify an assortment of processes of intense scientific interest, is nonetheless subject to intrinsic underestimation. A clever approach has evolved over several decades to more accurately estimate Φ Fm ' . The underlying rationale of the methodology requires a thorough and nuanced explanation, which is lacking in the literature. Herein, we systematically develop the essential rationale for accurately measuring Φ Fm ' based on the latest evolution of this approach, called multiphase flash (MPF) methodology.

Optimizing glutamine concentration enhances ex vivo expansion of natural killer cells through improved redox status.

Amino acids are vital components of the serum-free medium that influence the expansion and function of NK cells. This study aimed to clarify the relationship between amino acid metabolism and expansion and cytotoxicity of NK cells. Based on analyzing the mino acid metabolism of NK-92 cells and Design of Experiments (DOE), we optimized the combinations and concentrations of amino acids in NK-92 cells culture medium. The results demonstrated that NK-92 cells showed a pronounced demand for glutamine, serine, leucine, and arginine, in which glutamine played a central role. Significantly, at a glutamine concentration of 13 mM, NK-92 cells expansion reached 161.9 folds, which was significantly higher than 55.5 folds at 2.5 mM. Additionally, under higher glutamine concentrations, NK-92 cells expressed elevated levels of cytotoxic molecules, the level of cytotoxic molecules expressed by NK-92 cells was increased and the cytotoxic rate was 68.42%, significantly higher than that of 58.08% under low concentration. In view of the close relationship between glutamine metabolism and intracellular redox state, we investigated the redox status within the cells. This study demonstrated that intracellular ROS levels in higher glutamine concentrations were significantly lower than those under lower concentration cultures with decreased intracellular GSH/GSSG ratio, NADPH/NADP+ ratio, and apoptosis rate. These findings indicate that NK-92 cells exhibit improved redox status when cultured at higher glutamine concentrations. Overall, our research provides valuable insights into the development of serum-free culture medium for ex vivo expansion of NK-92 cells.