Cytochrome B Research Articles

NdhS interacts with cytochrome b6 f to form a complex in Arabidopsis.

Cyclic electron transport (CET) around photosystem I (PSI) is crucial for photosynthesis to perform photoprotection and sustain the balance of ATP and NADPH. However, the critical component of CET, cyt b6 f complex (cyt b6 f), functions in CET has yet to be understood entirely. In this study, we found that NdhS, a subunit of NADPH dehydrogenase-like (NDH) complex, interacted with cyt b6 f to form a complex in Arabidopsis. This interaction depended on the N-terminal extension of NdhS, which was conserved in eukaryotic plants but defective in prokaryotic algae. The migration of NdhS was much more in cyt b6 f than in PSI-NDH super-complex. Based on these results, we suggested that NdhS and NADP+ oxidoreductase provide a docking domain for the mobile electron carrier ferredoxin to transfer electrons to the plastoquinone pool via cyt b6 f in eukaryotic photosynthesis.

Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium.

Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b5M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.

Osmotic stress-induced localisation switch of CBR1 from mitochondria to the endoplasmic reticulum triggers ATP production via β-oxidation to respond to osmotic shock.

Drought and high salinity are major environmental factors that reduce plant growth and development, leading to loss of plant productivity in agriculture. Under these stress conditions, photosynthesis is greatly suppressed despite the high cellular energy cost of stress response processes. Currently, the process that allows plants to secure the energy required for osmotic stress responses remains elusive. Here, we provide evidence that cytochrome b5 reductase 1 (CBR1), a cytochrome b5 reductase, plays an important role in ATP production in response to NaCl and dehydration stresses. Overexpression and loss of function of CBR1 led to enhanced resistance and sensitivity, respectively, to osmotic stress. Upon exposure to osmotic stress, CBR1 was localised to the endoplasmic reticulum (ER) instead of to mitochondria, where it was localised under normal conditions. Transgenic plants overexpressing ER-targeted CBR1 showed enhanced resistance to osmotic stress. Moreover, CBR1-ER and CBR1-OX plants, had higher levels of ATP and unsaturated fatty acids under osmotic stress. However, these effects were abrogated by thioridazine and 2-deoxy glucose, inhibitors of β-oxidation and glycolysis, respectively. Based on these results, we propose that ER-localised CBR1 triggers ATP production via the production and β-oxidation of polyunsaturated fatty acids under osmotic stress.

High light-induced changes in whole-cell proteomic profile and its correlation with the organization of thylakoid super-complex in cyclic electron transport mutants of Chlamydomonas reinhardtii.

Light and nutrients are essential components of photosynthesis. Activating the signaling cascades is critical in starting adaptive processes in response to high light. In this study, we have used wild-type (WT), cyclic electron transport (CET) mutants like Proton Gradient Regulation (PGR) (PGRL1), and PGR5 to elucidate the actual role in regulation and assembly of photosynthetic pigment-protein complexes under high light. Here, we have correlated the biophysical, biochemical, and proteomic approaches to understand the targeted proteins and the organization of thylakoid pigment-protein complexes in the photoacclimation. The proteomic analysis showed that 320 proteins were significantly affected under high light compared to the control and are mainly involved in the photosynthetic electron transport chain, protein synthesis, metabolic process, glycolysis, and proteins involved in cytoskeleton assembly. Additionally, we observed that the cytochrome (Cyt) b6 expression is increased in the pgr5 mutant to regulate proton motive force and ATPase across the thylakoid membrane. The increased Cyt b6 function in pgr5 could be due to the compromised function of chloroplast (cp) ATP synthase subunits for energy generation and photoprotection under high light. Moreover, our proteome data show that the photosystem subunit II (PSBS) protein isoforms (PSBS1 and PSBS2) expressed more than the Light-Harvesting Complex Stress-Related (LHCSR) protein in pgr5 compared to WT and pgrl1 under high light. The immunoblot data shows the photosystem II proteins D1 and D2 accumulated more in pgrl1 and pgr5 than WT under high light. In high light, CP43 and CP47 showed a reduced amount in pgr5 under high light due to changes in chlorophyll and carotenoid content around the PSII protein, which coordinates as a cofactor for efficient energy transfer from the light-harvesting antenna to the photosystem core. BN-PAGE and circular dichroism studies indicate changes in macromolecular assembly and thylakoid super-complexes destacking in pgrl1 and pgr5 due to changes in the pigment-protein complexes under high light. Based on this study, we emphasize that this is an excellent aid in understanding the role of CET mutants in thylakoid protein abundances and super-complex organization under high light.

Pure total flavonoids from citrus alleviate oxidative stress and inflammation in nonalcoholic fatty liver disease by regulating the miR-137-3p/NOXA2/NOX2 pathway

BackgroundNonalcoholic fatty liver disease (NAFLD) has become a global health issue owing to its large disease population and high morbidity. We previously reported that the improvement in oxidative stress (OS) using pure total flavonoids from citrus (PTFC), flavonoids isolated from the peel of Citrus changshan-huyou Y.B. Chan, is a crucial strategy for NAFLD treatment. However, OS-associated intervention pathways in NAFLD remain unclear. MethodsIn this study, we used microRNA (miR)- and mRNA-sequencing to identify the pathway by which PTFC improve OS in NAFLD. Clinical data, mimic/inhibitor assays, and a dual-luciferase reporter assay were selected to verify the regulatory relationships of this pathway. Moreover, in vivo and in vitro experiments were used to confime the regulatory effect of PTFC on this pathway. ResultsmiR-seq, mRNA-seq, and bioinformatics analyses revealed that the miR-137–3p/neutrophil cytosolic factor 2 (NCF2, also known as NOXA2)/cytochrome b-245 beta chain (CYBB, also known as NOX2) pathway may be a target pathway for PTFC to improve OS and NAFLD. Additionally, bivariate logistic regression analysis combining the serum and clinical data of patients revealed NOX2 and NOXA2 as risk factors and total antioxidant capacity (indicator of OS level) as a protective factor for NAFLD. miR-137–3p mimic/inhibitor assays revealed that the upregulation of miR-137–3p is vital for improving cellular steatosis, OS, and inflammation. Dual-luciferase reporter assay confirmed that NOXA2 acts as an miR-137–3p sponge. These results co-determined that miR-137–3p/NOXA2/NOX2 is an essential pathway involved in NAFLD pathogenesis, including lipid accumulation, OS, and inflammation. In vivo and in vitro experiments further confirmed that the miR-137–3p/NOXA2/NOX2 pathway is regulated by PTFC. ConclusionPTFC alleviates OS and inflammation in NAFLD by regulating the miR-137–3p/NOXA2/NOX2 pathway.

A Novel Pathway of Functional microRNA Uptake and Mitochondria Delivery.

Extracellular microRNAs (miRNAs) play a critical role in horizontal gene regulation. Uptake of extracellular miRNAs by recipient cells and their intracellular transport, however, remains elusive. Here RNA phase separation is shown as a novel pathway of miRNA uptake. In the presence of serum, synthetic miRNAs rapidly self-assembly into ≈110nm discrete nanoparticles, which enable miRNAs' entry into different cells. Depleting serum cationic proteins prevents the formation of such nanoparticles and thus blocks miRNA uptake. Different from lipofectamine-mediated miRNA transfection in which majority of miRNAs are accumulated in lysosomes of transfected cells, nanoparticles-mediated miRNA uptake predominantly delivers miRNAs into mitochondria in a polyribonucleotide nucleotidyltransferase 1(PNPT1)-dependent manner. Functional assays further show that the internalized miR-21 via miRNA phase separation enhances mitochondrial translation of cytochrome b (CYB), leading to increase in adenosine triphosphate (ATP) and reactive oxygenspecies (ROS) reduction in HEK293T cells. The findings thus reveal a previously unrecognized mechanism for uptake and delivery functional extracellular miRNAs into mitochondria.

Far-red photosynthesis: Two charge separation pathways exist in plant Photosystem II reaction center

An alternative charge separation pathway in Photosystem II under the far-red light was proposed by us on the basis of electron transfer properties at 295 K and 5 K. Here we extend these studies to the temperature range of 77–295 K with help of electron paramagnetic resonance spectroscopy. Induction of the S2 state multiline signal, oxidation of Cytochrome b559 and ChlorophyllZ was studied in Photosystem II membrane preparations from spinach after application of a laser flashes in visible (532 nm) or far-red (730–750 nm) spectral regions. Temperature dependence of the S2 state signal induction after single flash at 730–750 nm (Tinhibition ~ 240 K) was found to be different than that at 532 nm (Tinhibition ~ 157 K). No contaminant oxidation of the secondary electron donors cytochrome b559 or chlorophyllZ was observed. Photoaccumulation experiments with extensive flashing at 77 K showed similar results, with no or very little induction of the secondary electron donors. Thus, the partition ratio defined as (yield of YZ/CaMn4O5-cluster oxidation):(yield of Cytb559/ChlZ/CarD2 oxidation) was found to be 0.4 at under visible light and 1.7 at under far-red light at 77 K. Our data indicate that different products of charge separation after far-red light exists in the wide temperature range which further support the model of the different primary photochemistry in Photosystem II with localization of hole on the ChlD1 molecule.

A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6 f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Oxidative Metabolism Genes in Ovarian Neoplasms

BACKGROUND: Reactive oxygen species play important and ambiguous role in carcinogenesis, and local oxidative metabolism may differ significantly from systemic metabolism and determine the processes occurring in tumor tissues.
 AIM: This study aimed to examine the expressions of key oxidative metabolism genes, particularly CYB5R, POR, NOX4, SOD1, NF-B, and NRF2, in ovarian neoplasm tissues, and determine cytochrome b5 reductase and cytochrome P450 reductase activity, blood neutrophil activity, and antioxidant indices in the blood plasma and peritoneal fluid.
 MATERIALS AND METHODS: The study included two groups of patients: a study group (n = 10) with ovarian adenocarcinoma and a comparison group (n = 6) with benign ovarian neoplasms. The expressions of CYB5R1, CYB5R2/R4, CYB5R3, POR, BIRC3, NOX4, NRF2, NF-B, SOD1, HMOX1, and BCL2 genes, cytochrome b5 reductase, and cytochrome P450 reductase activity, oxidative activity of blood neutrophils, and antioxidant potential of plasma and peritoneal fluid were evaluated in these two groups of women.
 RESULTS: The expression levels of CYB5R3 and BCL2 were significantly lower in adenocarcinoma tissues. The activities of cytochrome b5 reductase and cytochrome P450 reductase increased in the group with adenocarcinoma. On average, the activity of blood neutrophils corresponded to the reference values. For blood plasma, the antioxidant capacity were not different, whereas the antioxidant capacity in the peritoneal fluid increased approximately twofold in ovarian cancer.
 CONCLUSIONS: Significantly increased cytochrome b5 reductase activity in adenocarcinoma tissues may be a response to intracellular oxidative stress, whereas CYB5R3 gene expression may be reduced by a negative feedback mechanism. The activities of cytochrome P450 reductase and its gene change to a lesser extent in the presence of ovarian adenocarcinoma. The oxidative balance in the blood and peritoneal fluid correlated with the tissue expressions of NF-B and NRF2.

#2853 MITOCHONDRIAL DNA DEREGULATED PATTERN PARALLELS INFLAMMATION IN EARLY DIABETIC KIDNEY DISEASE OF TYPE 2 DIABETES MELLITUS PATIENTS

Abstract Background and Aims Mitochondrial DNA (mtDNA) abnormalities, as well as complex inflammatory processes involved in the pathogenesis of DKD may be detected early in the course of DKD. The aim of the study was to evaluate a potential relation of mtDNA modifications in blood and urine with podocyte injury and proximal tubule (PT) dysfunction in early DKD of type 2 diabetes mellitus (DM) patients. The hypothesis according to which mtDNA changes may be related to a specific inflammatory response in normoalbuminuric DKD of type 2 DM patients was also questioned. Method A total of 150 patients [52 patients with normoalbuminuria, 48 patients with microalbuminuria, and 50 patients with macroalbuminuria)] and 30 age- and gender-matched healthy controls were enrolled in this case series study. All patients were assessed concerning urinary albumin/creatinine ratio (UACR), biomarkers of podocyte damage (synaptopodin, podocalyxin) and of PT dysfunction (kidney injury molecule-1-KIM-1, N-acetyl-D-glucosaminidase-NAG), serum and urinary interleukins (IL-17A, IL-18, IL-10), and eGFR (creatinine-cystatin C). MtDNA-CN and nuclear DNA (nDNA) was quantified in peripheral blood and urine by qRT-PCR (ABI 7900-HT–Applied BioSystem). TaqMan assays were utilized for the assessment of cytochrome b (CYTB) gene, subunit 2 of NADH dehydrogenase (ND2), and of beta 2 microglobulin nuclear gene (B2M). mtDNA-CN was defined as the ratio of the number of mtDNA/nDNA copies, through analysis of the CYTB/B2M and ND2/B2M ratio. DNA standards were created by plasmidial cloning vectors. Cloning was verified by PCR sequencing and validation. Results Serum mtDNA was decreased, while urinary mtDNA was increased in all study groups as compared to healthy controls. In univariable linear analysis, serum mtDNA correlated directly with eGFR and negatively with UACR, urinary synaptopodin, urinary podocalyxin, urinary KIM-1, and urinary NAG. Also, serum mtDNA correlated negatively with serum IL-17A and urinary IL-17A, and with serum IL-18 and urinary IL-18, and directly with serum IL-10 and urinary IL-10. Urinary mtDNA correlated directly with UACR, synaptopodin, podocalyxin, KIM-1, NAG, IL-17A, and IL-18, and showed an inverse correlation with IL-10, and eGFR. Multivariable linear analysis provided models in which serum mtDNA correlated directly with IL-10, and indirectly with UACR, IL-17A, and KIM-1 (R2 = 0.626; p = 0.0001). Urinary mtDNA correlated directly with UACR, podocalyxin, IL-18, and NAG, and negatively with IL-10 and eGFR (R2 = 0.631; p = 0.0001). This data suggests that increased urinary mtDNA levels may be attributed to increased filtration of circulating DNA and that this may derive from filtered circulating mtDNA due to lesions to the glomerular and tubular structures. The correlations of mtDNA with proinflammatory cytokines IL-17A and IL-18, as well as with the anti-inflammatory cytokine IL-10 could explain the association of these cytokines with mtDNA variations and with the expression of podocyte damage and PT dysfunction biomarkers in all studied groups, even in normoalbuminuric type 2 DM patients. Conclusion MtDNA abnormalities and inflammatory processes occur early in the course of type 2 DM. MtDNA displays a specific profile in relation to inflammation both at podocyte and tubular level in normoalbuminuric patients with type 2 DM. These observations are independent of renal function decline.

Role of yceA-cybB-yceB operon in oxidative stress tolerance, swimming motility and antibiotic susceptibility of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is ubiquitous in the environment and is an important MDR opportunistic pathogen. Oxidative stress is an inevitable challenge to an aerobic bacterium. Accordingly, S. maltophilia has many capabilities to face variable oxidative stress. Some of the oxidative stress alleviation systems cross-protect bacteria from antibiotics. In our recent RNA-sequencing transcriptome analysis, we documented the increased expression of a three-gene cluster, yceA-cybB-yceB, in the presence of hydrogen peroxide (H2O2). The YceI-like, cytochrome b561 and YceI-like proteins encoded by yceA, cybB and yceB are located in the cytoplasm, inner membrane and periplasm, respectively. To characterize the role of the yceA-cybB-yceB operon of S. maltophilia in oxidative stress tolerance, swimming motility and antibiotic susceptibility. The presence of the yceA-cybB-yceB operon was verified by RT-PCR. The functions of this operon were revealed by in-frame deletion mutant construction and complementation assay. Expression of the yceA-cybB-yceB operon was assessed by quantitative RT-PCR. The yceA, cybB and yceB genes form an operon. Loss of function of the yceA-cybB-yceB operon compromised menadione tolerance, enhanced swimming motility and increased susceptibility to fluoroquinolone and β-lactam antibiotics. The expression of the yceA-cybB-yceB operon was up-regulated by oxidative stress, such as H2O2 and superoxide, and not impacted by antibiotics, such as fluoroquinolone and β-lactams. The evidence strongly supports the view that the physiological function of the yceA-cybB-yceB operon is to alleviate oxidative stress. The operon provides an additional example that oxidative stress alleviation systems can cross-protect S. maltophilia from antibiotics.

Insight into the molecular mechanism of phosphine toxicity provided by functional analysis of cytochrome b5 fatty acid desaturase and dihydrolipoamide dehydrogenase in the red flour beetle, Tribolium castaneum

Phosphine is the dominant chemical used in postharvest pest control. Widespread and highly frequent use of phosphine has been selected for pest insects, including Tribolium castaneum, which is highly resistant. Lipid peroxidation and reactive oxygen species (ROS) are two major factors determining phosphine toxicity; however, the mechanisms of production of these two factors in phosphine toxicity are still unknown. Here, we first determined the time course of phosphine-induced lipid peroxidation and ROS production in T. castaneum. Our results showed that lipid peroxidation occurs before ROS in the process of phosphine toxicity, and fumigated beetles with higher resistance levels were associated with weaker activity on lipid peroxidation and ROS. A significant decline in lipid peroxidation was observed in fumigated individuals after knockdown of cytochrome b5 fatty acid desaturase (Cyt-b5-r) via RNA interference (RNAi), indicating that Cyt-b5-r is critical for triggering phosphine-induced lipid peroxidation. Moreover, significant decreases in both ROS and mortality were detected in fumigated T. castaneum adults fed melatonin for 7 days, an inhibitor of lipid peroxidation. Cyt-b5-r RNAi also inhibited ROS production and mortality in phosphine-treated beetles. Meanwhile, a significant decrease in ROS production (68.4%) was detected in dihydrolipoamide dehydrogenase (DLD) knockdown individuals with phenotypes susceptible to phosphine, suggesting that lipid peroxidation initiates ROS with the expression of DLD. However, a significant increase in ROS (122.1%) was detected in the DLD knockdown beetles with strongly resistant phenotypes, indicating that the DLD-involved pathway may not be the only mechanism of ROS generation in phosphine toxicity and the existence of a moonlighting role in downregulating ROS in strongly resistant T. castaneum.

Role of the phospholipid binding sites, PX of p47phox and PB region of Rac1, in the formation of the phagocyte NADPH oxidase complex NOX2

In phagocytes, superoxide anion (O2−), the precursor of reactive oxygen species, is produced by the NADPH oxidase complex to kill pathogens. Phagocyte NADPH oxidase consists of the transmembrane cytochrome b558 (cyt b558) and four cytosolic components: p40phox, p47phox, p67phox, and Rac1/2. The phagocyte activation by stimuli leads to activation of signal transduction pathways. This is followed by the translocation of cytosolic components to the membrane and their association with cyt b558 to form the active enzyme.To investigate the roles of membrane-interacting domains of the cytosolic proteins in the NADPH oxidase complex assembly and activity, we used giant unilamellar phospholipid vesicles (GUV). We also used the neutrophil-like cell line PLB-985 to investigate these roles under physiological conditions. We confirmed that the isolated proteins must be activated to bind to the membrane. We showed that their membrane binding was strengthened by the presence of the other cytosolic partners, with a key role for p47phox. We also used a fused chimera consisting of p47phox(aa 1–286), p67phox(aa 1–212) and Rac1Q61L, as well as mutated versions in the p47phox PX domain and the Rac polybasic region (PB). We showed that these two domains have a crucial role in the trimera membrane-binding and in the trimera assembly to cyt b558. They also have an impact on O2.- production in vitro and in cellulo: the PX domain strongly binding to GUV made of a mix of polar lipids; and the PB region strongly binding to the plasma membrane of neutrophils and resting PLB-985 cells.

Metabolism of sphingadiene and characterization of the sphingadiene-producing enzyme FADS3

Of the long-chain bases (LCBs) that comprise the ceramides (CERs) present in mammals, only 4,14-sphingadiene (sphingadiene; SPD) has a cis double bond (at C14). Because of this unique structure, the metabolism of SPD may differ from that of other LCBs, but whether this is the case remains unclear. FADS3 is responsible for introducing the cis double bond in SPD. However, the substrate specificity of FADS3 and cofactors involved in the FADS3-catalyzed reaction are also unknown. In the present study, a cell-based assay using a ceramide synthase inhibitor and an in vitro experiment showed that FADS3 is active toward sphingosine (SPH)-containing CERs (SPH-CERs) but not toward free SPH. FADS3 exhibits specificity with respect to the chain length of the SPH moiety of SPH-CERs (active toward C16–20), but not that of the fatty acid moiety. Furthermore, FADS3 is active toward straight-chain and iso‐branched-chain SPH-containing CERs but not toward anteiso-branched forms. In addition to SPH-CERs, FADS3 also shows activity toward dihydrosphingosine-containing CERs, but this activity is approximately half of that toward SPH-CERs. It uses either NADH or NADPH as an electron donor, and the electron transfer is facilitated by cytochrome b5. The metabolic flow of SPD to sphingomyelin is predominant over that to glycosphingolipids. In the metabolic pathway from SPD to fatty acids, the chain length of the SPD is reduced by two carbons and the trans double bond at C4 is saturated. This study thus elucidates the enzymatic properties of FADS3 and the metabolism of SPD.

Methadone Reduced Nevirapine Pharmacokinetic Parameters in People Living With HIV in Malaysia

Introduction: The HIV epidemic in Malaysia predominantly affects males (90% of total HIV cases) mostly intravenous drugs users. Nevirapine-based of highly active antiretroviral therapy (HAART) once- or twice-daily dosage improve accessibility and effectiveness of antiretroviral treatment for HIV positive intravenous drug users (IDUs) receiving methadone maintenance treatment. Studies reported that concomitant administration of nevirapine with methadone reduced methadone plasma concentration. Since methadone and nevirapine were both known to be the substrate for cytochrome 2B6 (CYP 2B6), concomitant use of both drugs may affect nevirapine concentration too. However, methadone effect on nevirapine concentration is still unclear. This is a cross sectional study which reports how methadone co-administration affects the pharmacokinetic parameters of nevirapine in people living with HIV (PLHIV). Methods: 112 patients receiving nevirapine-based antiretroviral drugs were recruited. Seventeen were maintained with methadone without withdrawal symptoms. High-performance liquid chromatography was used to measure plasma nevirapine concentrations. Nevirapine population pharmacokinetics was modelled with a non-parametric approach using Pmetrics software. Result: According to univariate analysis, concurrent methadone administration increased the clearance of nevirapine by 25.3% (p = 0.046). Multivariate analysis showed that methadone medication was independently linked with lower nevirapine concentrations and area-under-curve (Cmin was reduced by 15.2%, p = 0.011, Cmax 19.5%; p = 0.003, AUC12 16.2%; p = 0.021 respectively). Conclusion: This study provides in-vivo evidence of methadone co-administration reducing nevirapine exposure. Since a low concentration of nevirapine will lead to treatment failure, monitoring is essential for PLHIV using both medications at the same time.

Corncob bedding alters fasting blood glucose and vascular function in mice

Rodent husbandry requires careful consideration of environmental factors that may impact colony performance and subsequent physiological studies. Of note, recent reports have suggested corncob bedding is potentially a confounding variable affecting a broad range of organ systems. As corncob bedding may contain digestible hemicelluloses, trace sugars, and fiber, we hypothesized that corncob bedding impacts murine vascular function and overnight fasting blood glucose. Here, we compared mice housed on corncob bedding, which were then fasted overnight on either corncob or ALPHA-dri bedding, a paper cellulose alternative. Male and female mice were used from two non-induced, endothelial-specific conditional knock-out strains (Cadherin 5-CreERT2, floxed hemoglobin alpha 1 [Hba1] or Cadherin 5-CreERT2, floxed cytochrome B5 reductase 3 [CyB5R3]) on a C57BL/6J genetic background. After fasting overnight, initial blood glucose was measured, and mice were anesthetized with isoflurane for measurement of blood perfusion via laser speckle contrast analysis using a PeriMed PeriCam PSI NR system. After a 15-minute equilibration, the mice were injected intraperitoneally with phenylephrine (5 mg/kg) or saline and monitored for changes in blood perfusion. After a 15-minute response period, blood glucose was remeasured post-procedure. Consistently, mice fasted on corncob bedding had higher blood glucose than the ALPHA-dri group. In the CyB5R3 strain, corncob bedding caused a reduction in phenylephrine-mediated vasoconstriction. In the Hba1 strain, phenylephrine-induced vasoconstriction was more variable in the corncob group. Altogether, this work suggests that corncob bedding could be a confounding variable for vascular measurements and fasting blood glucose in mice. To promote scientific rigor and improve reproducibility, bedding type should be included in published methods. NIH R01HL155618 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Development of Multiplex PCR and Melt-Curve Analysis for the Molecular Identification of Four Species of the Mullidae Family, Available in the Market.

The authentication of food products and the verification of their identity are of major importance for consumers. Food fraud through mislabeling is an illegal practice consisting of the substitution of an expensive food product by a relatively cheaper one, misleading false labelling of their origin and adulteration in processed or frozen products. This issue is particularly of high importance concerning fish and seafood, which are easily adulterated primarily due to difficult morphological identification. Fish species of the Mullidae family are considered among the most high-valued seafood products traded in Greece and Eastern Mediterranean in general, in terms of the price and demand. Specifically, the red mullet (Mullus barbatus) and the striped red mullet (Mullus surmuletus) are both indigenous in the Aegean (FAO Division 37.3.1) and the Ionian (FAO Division 37.2.2) Seas, with high levels of consumers' preferences. However, they could be easily adulterated or misidentified by the invasive Aegean Sea Lessepsian migrator goldband goatfish (Upeneus moluccensis) as well as by the imported West African goatfish (Pseudupeneus prayensis). Keeping this in mind, we designed two novel, time-saving and easy-to-apply multiplex PCR assays and one multiple Melt-Curve analysis real-time PCR for the identification of these four species. These methodologies are based on species-specific primers targeting single nucleotide polymorphisms (SNPs) detected via sequencing analysis of the mitochondrial cytochrome C oxidase subunit I (CO1) and of the cytochrome b (CYTB) genes in newly collected individuals, with additional comparison with congeneric and conspecific haplotypes obtained from the GenBank database. Both methodologies, targeting CO1 or CYTB, utilize one common and four diagnostic primers, producing amplicons of different length that are easily and reliably separated on agarose gel electrophoresis, yielding a single clear band of diagnostic size for each species or a certain Melt-Curve profile. The applicability of this cost-effective and fast methodology was tested in 328 collected specimens, including 10 cooked samples obtained from restaurants. In the vast majority (327 out of the 328) of the specimens tested, one single band was produced, in agreement with the expected products with a single exception a M. barbatus sample that was identified as M. surmuletus, the identity of which was confirmed using sequencing, indicating erroneous morphological identification. The developed methodologies are expected to contribute to the detection of commercial fraud in fish authentication.

STN7 is not essential for developmental acclimation of Arabidopsis to light intensity.

Plants respond to changing light intensity in the short term through regulation of light harvesting, electron transfer, and metabolism to mitigate redox stress. A sustained shift in light intensity leads to a long-term acclimation response (LTR). This involves adjustment in the stoichiometry of photosynthetic complexes through de novo synthesis and degradation of specific proteins associated with the thylakoid membrane. The light-harvesting complex II (LHCII) serine/threonine kinase STN7 plays a key role in short-term light harvesting regulation and was also suggested to be crucial to the LTR. Arabidopsis plants lacking STN7 (stn7) shifted to low light experience higher photosystem II (PSII) redox pressure than the wild type or those lacking the cognate phosphatase TAP38 (tap38), while the reverse is true at high light, where tap38 suffers more. In principle, the LTR should allow optimisation of the stoichiometry of photosynthetic complexes to mitigate these effects. We used quantitative label-free proteomics to assess how the relative abundance of photosynthetic proteins varied with growth light intensity in wild-type, stn7, and tap38 plants. All plants were able to adjust photosystem I, LHCII, cytochrome b6 f, and ATP synthase abundance with changing white light intensity, demonstrating neither STN7 nor TAP38 is crucial to the LTR per se. However, stn7 plants grown for several weeks at low light (LL) or moderate light (ML) still showed high PSII redox pressure and correspondingly lower PSII efficiency, CO2 assimilation, and leaf area compared to wild-type and tap38 plants, hence the LTR is unable to fully ameliorate these symptoms. In contrast, under high light growth conditions the mutants and wild type behaved similarly. These data are consistent with the paramount role of STN7-dependent LHCII phosphorylation in tuning PSII redox state for optimal growth in LL and ML conditions.

Effect of the Anticancer Drug Doxorubicin (Adriamycin) on Antioxidant Studies and Ultrastructural Investigation in the Liver, Kidney, and Heart Tissues of Male Rats

Doxorubicin is a well-known antineoplastic agent that has proved to be successful in the treatment of various types of cancer. I used rats as the model to evaluate the effect of doxorubicin on antioxidant studies and ultrastructural investigations in the liver, kidney, and heart tissues. Male albino rats were given 1.0 mg/kg body weight of the anticancer drug doxorubicin intraperitoneally three times a week for 52 days. This was for a total of 18 doses. Control animals received 52 doses of 0.5 ml of saline over 52 days. The body weights of rats injected with doxorubicin experienced a significant decrease after the last dose compared to the control group of rats. In this study, the weights of the heart, kidneys, and liver were measured. Except for cardiac tissues, the protein content in the aforementioned tissues in treated rats was significantly different from the control. Glutathione (GSH) levels in the kidneys of experimental rats were not significantly lower (7.946 ± 0.781) compared to controls (8.06 ± 0.74) but there was a non-significant increase in GSH levels in the liver (17.095 ± 1.066) compared to controls (13.8 ± 1.3). In addition, the mean GSH levels in doxorubicin-treated hearts were significantly lower (7.9462 ± 0.781) compared to controls (8.06 ± 0.74). Lipid peroxidation (Lpx) and malondialdehyde content (MDA), a marker of lipid peroxidation, were found in much lower concentrations in the liver organ of the doxorubicin-treated group (0.0162 ± 0.00086) as compared to (0.20 ± 0.02) controls, and MDA content in the kidney was decreased (0.0239 ± 0.0003) compared to control rats (0.31 ± 0.03), as well as heat production (0.0398 ± 0.00097) compared to (47.451 ± 1.708) controls. Glutathione reductase (GR) levels were significantly elevated in the same tissue treatment group. Glutathione-S-Transferase (G-S-T) activity was assessed and significantly increased in all tissues in the doxorubicin model. Glutathione peroxidase (GPx) activity showed a significant decrease in all the above tissues after doxorubicin injection. The catalase (CAT) activity of doxorubicin was greatly increased in one treated rat. In the doxorubicin-treated group, levels of cytochrome p450 (CYTp450) were significantly decreased in liver and kidney tissue and significantly elevated in heart tissue. After doxorubicin treatment, cytochrome b5 (CYTb5) levels in liver tissues increased significantly (837.177± 61.197) compared to controls (615 ± 37.0), and the contents of cytochrome b5 in rats' kidneys increased significantly (447.685 ± 35.215) compared to controls (2605.5± 259.2). and cytochrome b5 in heart tissues was lower (165.352± 8.7) when compared to controls (88± 0.4). The results showed that there were few obvious changes in histological, ultrastructural, and biochemical changes in liver tissue in the doxorubicin model. Long-term doxorubicin treatment in kidney tissue results in no significant changes at the light microscopic level, but the electron microscopic level reveals no change from a histological point of view.

OsCYBDOMG1, a cytochrome b561 domain-containing protein, regulates salt tolerance and grain yield in rice

OsCYBDOMG1 positively regulates salt tolerance, plant growth, and grain yield by affecting ascorbate biosynthesis and redox state. Soil salinity is a major abiotic stress affecting rice growth and productivity. Many genes involved in the salt stress response have been identified, but the precise mechanisms underlying salt tolerance remain unclear. In this study, we isolated a salt-sensitive mutant of rice, rss5, which exhibited more severe wilting and chlorosis with a significant increase in lipid peroxidation, electrolyte leakage, and shoot Na+ concentration compared to wild-type plants. Map-based cloning, MutMap analysis, and genetic complementation revealed that a single-nucleotide mutation in a gene encoding a cytochrome b561 domain-containing protein (OsCYBDOMG1) was responsible for the mutant phenotype of rss5. The OsCYBDOMG1 gene was mainly expressed in young shoots and nodes, and the encoded protein was principally located in the plasma membrane and endoplasmic reticulum. Mutations of OsCYBDOMG1 resulted in decreased ascorbic acid (AsA) content and AsA/DHA (dehydroascorbate) ratio, which led to increased H2O2 accumulation and reduced salt tolerance. Moreover, plant growth and grain yield of rss5 and the OsCYBDOMG1 knockout mutant (cr-1) were significantly decreased compared to wild-type plants under normal conditions. The elite haplotype of OsCYBDOMG1 associated with higher salt tolerance and grain width and weight was mainly existed in japonica varieties. These results suggest that OsCYBDOMG1 plays an important role in the regulation of salt tolerance, plant growth, and grain yield in rice.