Mapping of oxidative modifications on the alpha-keto glutarate dehydrogenase complex induced by singlet oxygen: effects on structure and activity.

The large multi-subunit mitochondrial alpha-keto glutarate dehydrogenase (KGDH) complex plays a key, rate-determining, role in the tricarboxylic acid (Krebs) cycle, catalyzing the conversion of alpha-keto glutarate to succinyl-CoA. This complex is both a source and target of oxidants, but the sites of modification and association with structural changes and activity loss are poorly understood. We report here oxidative modifications induced by Rose Bengal (RB) in the presence of O2, a source of singlet oxygen (1O2). A rapid loss of activity was detected, with this being dependent on light exposure, illumination time, and the presence of RB and O2. Activity loss was enhanced by D2O (consistent with 1O2 involvement), but diminished by both pre- and (to a lesser extent) post-illumination addition of lipoic acid and lipoamide. Aggregates containing all three KGDH subunits were detected on photooxidation. LC-MS experiments provided evidence for oxidation at 45 sites, including specific Met, His, Trp, Tyr residues and the lipoyllysine active-site cofactor. Products include mono- and di-oxygenated species, and kynurenine from Trp. Mapping of the modifications to the 3-D structure showed that these are localized to both the inner channel and the external surface, consistent with reactions of free 1O2, however the sites and extent of modification do not correlate with their solvent accessibility. These products are generated concurrently with loss of activity, indicative of strong links between these events. These data provide evidence for the impairment of KGDH activity by 1O2 via the oxidation of specific residues on the protein subunits of the complex.

Concurrent use of hormonal long-acting reversible contraception by women of reproductive age dispensed teratogenic medications, Australia, 2013-2021: a retrospective cohort study.

OBJECTIVES: To examine patterns in the dispensing of category X medications (Therapeutic Goods Administration categorisation system for prescribing medicines in pregnancy) to women aged 15-49 years in Australia during 2008-2021, and patterns of concurrent use of hormonal long-acting reversible contraception (LARC) and other hormonal contraception. STUDY DESIGN: Retrospective cohort study; analysis of 10% random sample of national Pharmaceutical Benefits Scheme dispensing data. PARTICIPANTS, SETTING: Women aged 15-49 years dispensed category X medications, Australia, 1 January 2013 - 31 December 2021. MAIN OUTCOME MEASURES: Incident and prevalent dispensing of category X medications, by medication class, age group, and year; contraceptive overlap (proportions of women dispensed hormonal LARC or other hormonal contraception that overlapped the first dispensing of category X medications), by medication class. RESULTS: Among 15 627 women aged 15-49 years dispensed category X medications during 2013-2021, the prevalence of dispensing increased from 4.6 in 2013 to 8.7 per 1000 women aged 15-49 years in 2021; the largest increase was for the dispensing of dermatological agents, from 3.9 to 7.9 per 1000 women aged 15-49 years. LARC overlap was inferred for 2059 women at the time of first dispensing of category X medications (13.2%); 3441 had been dispensed any type of hormonal contraception (22.1%). The proportion with LARC overlap was smallest for those dispensed dermatological agents (1806 of 14 331 women, 12.6%); for this drug class, both LARC overlap (adjusted odds ratio [aOR], 0.17; 95% confidence interval [CI], 0.14-0.20) and any hormonal contraception overlap (aOR, 0.28; 95% CI, 0.25-0.32) were less likely for those aged 15-19 years than for women aged 25-29 years. CONCLUSIONS: Concurrent use of highly effective hormonal contraception at the time of first dispensing of category X medications is low in Australia, raising concerns about potential fetal harms during unintended pregnancies. Awareness of the importance of hormonal contraception and its uptake by women prescribed category X medications should be increased.

Maternal modifiable factors and risk of congenital heart defects: systematic review and causality assessment.

OBJECTIVE: Primary prevention strategies are critical to reduce the global burden of congenital heart defects (CHDs); this requires robust knowledge of causal agents. We aimed to review associations between CHDs and maternal advanced age, obesity, diabetes, hypertension, smoking and alcohol consumption and assess the causal nature of the associations. DESIGN: Systematic review of reviews with application of a Bradford Hill criteria score-based causal assessment system. DATA SOURCES: We searched PubMed, Embase and Episteminokos (January 1990-April 2023). ELIGIBILITY CRITERIA: Systematic reviews of original epidemiological studies reporting association (relative risk) between one or more of the above maternal factors and CHDs overall (any type) in subsequent offspring. DATA EXTRACTION AND SYNTHESIS: Two independent reviewers selected eligible reviews, assessed the risk of bias and assigned the strength of evidence for causality. RESULTS: There was strong evidence of a causal relationship between CHDs and maternal obesity (prepregnancy and early pregnancy) and pre-existing diabetes (six of seven Bradford Hill criteria met). For pre-existing hypertension (strength and biological gradient not met), and advanced age (strength, consistency and biological gradient not met), causal evidence was moderate. Evidence for the causal contribution of gestational diabetes, gestational hypertension, smoking and alcohol consumption was weak (strength, consistency, temporality and biological gradient not met). CONCLUSIONS: CHDs can be reduced with stronger action to reduce maternal obesity and pre-existing diabetes prevalence. Investigating environmental exposures that have received limited attention, such as air pollutants and chemical exposures, is important to further inform prevention.

Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production.

The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.

Modification of extracellular matrix proteins by oxidants and electrophiles.

The extracellular matrix (ECM) is critical to biological architecture and determines cellular properties, function and activity. In many situations it is highly abundant, with collagens and elastin being some of the most abundant proteins in mammals. The ECM comprises of multiple different protein species and sugar polymers, with both different isoforms and post-translational modifications (PTMs) providing a large variety of microenvironments that play a key role in determining tissue structure and health. A number of the PTMs (e.g. cross-links) present in the ECM are critical to integrity and function, whereas others are deleterious to both ECM structure and associated cells. Modifications induced by reactive oxidants and electrophiles have been reported to accumulate in some ECM with increasing age. This accumulation can be exacerbated by disease, and in particular those associated with acute or chronic inflammation, obesity and diabetes. This is likely to be due to higher fluxes of modifying agents in these conditions. In this focused review, the role and effects of oxidants and other electrophiles on ECM are discussed, with a particular focus on the artery wall and atherosclerotic cardiovascular disease. Modifications generated on ECM components are reviewed, together with the effects of these species on cellular properties including adhesion, proliferation, migration, viability, metabolic activity, gene expression and phenotype. Increasing data indicates that ECM modifications are both prevalent in human and mammalian tissues and play an important role in disease development and progression.

Elevated levels of iodide promote peroxidase-mediated protein iodination and inhibit protein chlorination.

At inflammatory sites, immune cells generate oxidants including H2O2. Myeloperoxidase (MPO), released by activated leukocytes employs H2O2 and halide/pseudohalides to form hypohalous acids that mediate pathogen killing. Hypochlorous acid (HOCl) is a major species formed. Excessive or misplaced HOCl formation damages host tissues with this linked to multiple inflammatory diseases. Previously (Redox Biology, 2020, 28, 101331) we reported that iodide (I⁻) modulates MPO-mediated protein damage by decreasing HOCl generation with concomitant hypoiodous acid (HOI) formation. HOI may however impact on protein structure, so in this study we examined whether and how HOI, from peroxidase/H2O2/I⁻ systems ± Cl⁻, modifies proteins. Experiments employed MPO and lactoperoxidase (LPO) and multiple proteins (serum albumins, anastellin), with both chemical (intact protein and peptide mass mapping, LC-MS) and structural (SDS-PAGE) changes assessed. LC-MS analyses revealed dose-dependent iodination of anastellin and albumins by LPO/H2O2 with increasing I⁻. Incubation of BSA with MPO/H2O2/Cl⁻ revealed modest chlorination (Tyr286, Tyr475, ∼4 %) and Met modification. Lower levels of these species, and extensive iodination at specific Tyr and His residues (>20 % modification with ≥10 µM I⁻) were detected with increasing I⁻. Anastellin dimerization was inhibited by increasing I⁻, but less marked changes were observed with albumins. These data confirm that I⁻ competes with Cl⁻ for MPO and is an efficient HOCl scavenger. These processes decrease protein chlorination and oxidation, but result in extensive iodination. This is consistent with published data on the presence of iodinated Tyr on neutrophil proteins. The biological implications of protein iodination relative to chlorination require further clarification.

Clomiphene Citrate Administered in Periconception Phase Causes Fetal Loss and Developmental Impairment in Mice.

Clomiphene citrate is a common treatment for ovulation induction in subfertile women, but its use is associated with elevated risk of adverse perinatal outcomes and birth defects. To investigate the biological plausibility of a causal relationship, this study investigated the consequences in mice for fetal development and pregnancy outcome of periconception clomiphene citrate administration at doses approximating human exposures. A dose-dependent adverse effect of clomiphene citrate given twice in the 36 hours after mating was seen, with a moderate dose of 0.75 mg/kg sufficient to cause altered reproductive outcomes in 3 independent cohorts. Viable pregnancy was reduced by 30%, late gestation fetal weight was reduced by 16%, and ∼30% of fetuses exhibited delayed development and/or congenital abnormalities not seen in control dams, including defects of the lung, kidney, liver, eye, skin, limbs, and umbilicus. Clomiphene citrate also caused a 30-hour average delay in time of birth, and elevated rate of pup death in the early postnatal phase. In surviving offspring, growth trajectory tracking and body morphometry analysis at 20 weeks of age showed postweaning growth and development similar to controls. A dysregulated inflammatory response in the endometrium was observed and may contribute to the underlying pathophysiological mechanism. These results demonstrate that in utero exposure to clomiphene citrate during early pregnancy can compromise implantation and impact fetal growth and development, causing adverse perinatal outcomes. The findings raise the prospect of similar iatrogenic effects in women where clomiphene citrate may be present in the periconception phase unless its use is well-supervised.

Beta-Hydroxybutyrate Levels and Risk of Diabetic Ketoacidosis in Adults with Type 1 Diabetes Treated with Sotagliflozin.

Introduction: Sodium glucose cotransporter inhibitors may increase beta-hydroxybutyrate (BHB) in insulin-requiring patients. We determined factors associated with BHB changes from baseline (ΔBHB) and diabetic ketoacidosis (DKA) in patients with type 1 diabetes (T1D) receiving sotagliflozin as an insulin adjunct. Research Design and Methods: This post hoc analysis compared ΔBHB levels in adults with T1D receiving sotagliflozin 400 mg or placebo for 6 months. We evaluated clinical and metabolic factors associated with ΔBHB and used logistic regression models to determine predictors associated with BHB values >0.6 and >1.5 mmol/L (inTandem3 population; N = 1402) or with DKA events in a pooled analysis (inTandem1-3; N = 2453). Results: From baseline (median, 0.13 mmol/L), median fasting BHB increased by 0.04 mmol/L (95% confidence interval, 0.03-0.05; P < 0.001) at 24 weeks with sotagliflozin versus placebo; 67% of patients had no or minimal changes in BHB over time. Factors associated with on-treatment BHB >0.6 or >1.5 mmol/L included baseline BHB and sotagliflozin use. Age, insulin pump use, sotagliflozin use, baseline BHB, and ΔBHB were significantly associated with DKA episodes. Independent of treatment, DKA risk increased by 18% with each 0.1-mmol/L increase in baseline BHB and by 8% with each 0.1-mmol/L increase from baseline. Conclusion: Incremental increases in baseline BHB and ΔBHB were associated with a higher DKA risk independent of treatment. Adding sotagliflozin to insulin increased median BHB over 24 weeks in patients with T1D and was associated with increased DKA events. These results highlight the importance of BHB testing and monitoring and individualizing patient education on DKA risk, mitigation, identification, and treatment.

Sotagliflozin and Kidney Outcomes, Kidney Function, and Albuminuria in Type 2 Diabetes and CKD: A Secondary Analysis of the SCORED Trial.

BACKGROUND: In the initial analysis of the Effect of Sotagliflozin on Cardiovascular and Renal Events in Patients with Type 2 Diabetes and Moderate Renal Impairment Who Are at Cardiovascular Risk (SCORED) trial, because of early trial termination and suspension of adjudication, reconciliation of eGFR laboratory data and case report forms had not been completed. This resulted in a small number of kidney composite events and a nominal effect of sotagliflozin versus placebo on this outcome. This exploratory analysis uses laboratory eGFR data, regardless of case report form completion, to assess the effects of sotagliflozin on the predefined kidney composite end point in the SCORED trial and additional cardiorenal composite end points. METHODS: SCORED was a multicenter, randomized trial evaluating cardiorenal outcomes with sotagliflozin versus placebo in 10,584 patients with type 2 diabetes and CKD. This exploratory analysis used laboratory data to derive the eGFR components and case report form data for the non-laboratory-defined components that together made up the kidney and cardiorenal composites. AKI was also assessed in this dataset. RESULTS: Using laboratory data, 223 events were identified, and sotagliflozin reduced the risk of the composite of first event of sustained ≥50% decline in eGFR, eGFR <15 ml/min per 1.73 m 2 , dialysis, or kidney transplant with 87 events (1.6%) in the sotagliflozin group and 136 events (2.6%) in the placebo group (hazard ratio [95% confidence interval], 0.62 [0.48 to 0.82]), P < 0.001). Sotagliflozin reduced the risk of a cardiorenal composite end point defined as the abovementioned composite plus cardiovascular or kidney death with 239 events (4.5%) in the sotagliflozin group and 306 events (5.7%) in the placebo group (hazard ratio [95% confidence interval], 0.77 [0.65 to 0.91], P = 0.0023). The results were consistent when using different eGFR decline thresholds and when only including kidney death in composites (all P < 0.01). The incidence of AKI was similar between treatment groups. CONCLUSIONS: In this exploratory analysis using the complete laboratory dataset, sotagliflozin reduced the risk of kidney and cardiorenal composite end points in patients with type 2 diabetes and CKD. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT03315143 .

Proteomic analysis of the extracellular matrix of human atherosclerotic plaques shows marked changes between plaque types.

Cardiovascular disease is the leading cause of death, with atherosclerosis the major underlying cause. While often asymptomatic for decades, atherosclerotic plaque destabilization and rupture can arise suddenly and cause acute arterial occlusion or peripheral embolization resulting in myocardial infarction, stroke and lower limb ischaemia. As extracellular matrix (ECM) remodelling is associated with plaque instability, we hypothesized that the ECM composition would differ between plaques. We analyzed atherosclerotic plaques obtained from 21 patients who underwent carotid surgery following recent symptomatic carotid artery stenosis. Plaques were solubilized using a new efficient, single-step approach. Solubilized proteins were digested to peptides, and analyzed by liquid chromatography-mass spectrometry using data-independent acquisition. Identification and quantification of 4498 plaque proteins was achieved, including 354 ECM proteins, with unprecedented coverage and high reproducibility. Multidimensional scaling analysis and hierarchical clustering indicate two distinct clusters, which correlate with macroscopic plaque morphology (soft/unstable versus hard/stable), ultrasound classification (echolucent versus echogenic) and the presence of hemorrhage/ulceration. We identified 714 proteins with differential abundances between these groups. Soft/unstable plaques were enriched in proteins involved in inflammation, ECM remodelling, and protein degradation (e.g. matrix metalloproteinases, cathepsins). In contrast, hard/stable plaques contained higher levels of ECM structural proteins (e.g. collagens, versican, nidogens, biglycan, lumican, proteoglycan 4, mineralization proteins). These data indicate that a single-step proteomics method can provide unique mechanistic insights into ECM remodelling and inflammatory mechanisms within plaques that correlate with clinical parameters, and help rationalize plaque destabilization. These data also provide an approach towards identifying biomarkers for individualized risk profiling of atherosclerosis.

Oxidation of the active site cysteine residue of glyceraldehyde-3-phosphate dehydrogenase to the hyper-oxidized sulfonic acid form is favored under crowded conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key cellular enzyme, with major roles in both glycolysis, and 'moonlighting' activities in the nucleus (uracil DNA glycosylase activity, nuclear protein nitrosylation), as a regulator of mRNA stability, a transferrin receptor, and as an antimicrobial agent. These activities are dependent, at least in part, on the integrity of an active site Cys residue, and a second neighboring Cys. These residues are differentially sensitive to oxidation, and determine both its catalytic activity and the redox signaling capacity of the protein. Such Cys modification is critical to cellular adaptation to oxidative environments by re-routing metabolic pathways to favor NADPH generation and antioxidant defenses. Despite the susceptibility of GAPDH to oxidation, it remains a puzzle as to how this enzyme acts as a redox signaling hub for oxidants such as hydrogen peroxide (H2O2) in the presence of high concentrations of specialized high-efficiency peroxide-removing enzymes. One possibility is that crowded environments, such as the cell cytosol, alter the oxidation pathways of GAPDH. In this study, we investigated the role of crowding (induced by dextran) on H2O2- and SIN-1-induced GAPDH oxidation, with data for crowded and dilute conditions compared. LC-MS/MS data revealed a lower extent of modification of the catalytic Cys under crowded conditions (i.e. less monomer units modified), but enhanced formation of the sulfonic acid resulting from hyper-oxidation. This effect was not observed with SIN-1. These data indicate that molecular crowding can modulate the oxidation pathways of GAPDH and its extent of oxidation and inactivation.

Efficacy of Sotagliflozin in Adults With Type 2 Diabetes in Relation to Baseline Hemoglobin A1c.

BACKGROUND: The SCORED (Effect of Sotagliflozin on Cardiovascular and Renal Events in Patients with Type 2 Diabetes and Moderate Renal Impairment Who Are at Cardiovascular Risk) and SOLOIST-WHF (Effect of Sotagliflozin on Cardiovascular Events in Patients with Type 2 Diabetes Post Worsening Heart Failure) trials demonstrated that sotagliflozin, an SGLT1 and SGLT2 inhibitor, improves outcomes in individuals with type 2 diabetes who have heart failure (HF) or kidney disease. OBJECTIVES: We assessed the efficacy of sotagliflozin on HF clinical outcomes in individuals with differing baseline glycosylated hemoglobin (HbA1c) levels. METHODS: We included all adults from SCORED and SOLOIST-WHF. The primary outcome was a composite of cardiovascular death, hospitalizations for HF, and urgent visits for HF. The efficacy of sotagliflozin compared with placebo was evaluated by baseline HbA1c using competing-risk marginal proportional hazards models. RESULTS: We identified 11,744 adults. Individuals with HbA1c ≤7.5% experienced the primary outcome at a lower rate in the sotagliflozin group (11.2 per 100 person-years) than the placebo group (15.5 per 100 person-years) (HR: 0.73; 95% CI: 0.57-0.93). Similarly, individuals with HbA1c of 7.6% to 9.0% experienced the primary outcome at a lower rate in the sotagliflozin group (7.3 per 100 person-years) than the placebo group (9.4 per 100 person-years) (HR: 0.77; 95% CI: 0.63-0.96). These findings were also consistent among individuals with HbA1c >9.0%, with a primary outcome rate in the sotagliflozin group (7.8 per 100 person-years) that was lower than the placebo group (11.6 per 100 person-years) (HR: 0.65; 95% CI: 0.50-0.84). The efficacy of sotagliflozin was consistent by baseline HbA1c level (P for interaction = 0.58). CONCLUSIONS: In individuals with type 2 diabetes and either HF or kidney disease, sotagliflozin reduced HF outcomes irrespective of baseline HbA1c.

The enzymes of the oxidative phase of the pentose phosphate pathway as targets of reactive species: consequences for NADPH production.

The pentose phosphate pathway (PPP) is a key metabolic pathway. The oxidative phase of this process involves three reactions catalyzed by glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydrogenase (6PGDH) enzymes. The first and third steps (catalyzed by G6PDH and 6PGDH, respectively) are responsible for generating reduced nicotinamide adenine dinucleotide phosphate (NAPDH), a key cofactor for maintaining the reducing power of cells and detoxification of both endogenous and exogenous oxidants and electrophiles. Despite the importance of these enzymes, little attention has been paid to the fact that these proteins are targets of oxidants. In response to oxidative stimuli metabolic pathways are modulated, with the PPP often up-regulated in order to enhance or maintain the reductive capacity of cells. Under such circumstances, oxidation and inactivation of the PPP enzymes could be detrimental. Damage to the PPP enzymes may result in a downward spiral, as depending on the extent and sites of modification, these alterations may result in a loss of enzymatic activity and therefore increased oxidative damage due to NADPH depletion. In recent years, it has become evident that the three enzymes of the oxidative phase of the PPP have different susceptibilities to inactivation on exposure to different oxidants. In this review, we discuss existing knowledge on the role that these enzymes play in the metabolism of cells, and their susceptibility to oxidation and inactivation with special emphasis on NADPH production. Perspectives on achieving a better understanding of the molecular basis of the oxidation these enzymes within cellular environments are given.

Genotoxicity assessment of 1,4-anhydro-4-seleno-D-talitol (SeTal) in human liver HepG2 and HepaRG cells.

1,4-Anhydro-4-seleno-D-talitol (SeTal) is a highly water-soluble selenosugar with interesting antioxidant and skin-tissue-repair properties; it is highly stable in simulated gastric and gastrointestinal fluids and is a potential pharmaceutical ingredient that may be administered orally. Hepatic toxicity is often a major problem with novel drugs and can result in drug withdrawal from the market. Predicting hepatotoxicity is therefore essential to minimize late failure in the drug-discovery process. Herein, we report in vitro studies to evaluate the cytotoxic and genotoxic potential of SeTal in HepG2 and hepatocyte-like differentiated HepaRG cells. Except for extremely high concentrations (10 mM, 68 h-treatment in HepG2), SeTal did not affect the viability of each cell type. While the highest examined concentrations (0.75 and 1 mM in HepG2; 1 mM in HepaRG) were observed to induce primary DNA damage, SeTal did not exhibit clastogenic or aneugenic activity toward either HepG2 or HepaRG cells. Moreover, no significant cytostasis variations were observed in any experiment. The clearly negative results observed in the CBMN test suggest that SeTal might be used as a potential active pharmaceutical ingredient. The present study will be useful for the selection of non-toxic concentrations of SeTal in future investigations.

Recent Advances in the Synthesis and Antioxidant Activity of Low Molecular Mass Organoselenium Molecules.

Selenium is an essential trace element in living organisms, and is present in selenoenzymes with antioxidant activity, like glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). The search for small selenium-containing molecules that mimic selenoenzymes is a strong field of research in organic and medicinal chemistry. In this review, we review the synthesis and bioassays of new and known organoselenium compounds with antioxidant activity, covering the last five years. A detailed description of the synthetic procedures and the performed in vitro and in vivo bioassays is presented, highlighting the most active compounds in each series.

Detrimental Actions of Chlorinated Nucleosides on the Function and Viability of Insulin-Producing Cells.

Neutrophils are innate immune cells that play a key role in pathogen clearance. They contribute to inflammatory diseases, including diabetes, by releasing pro-inflammatory cytokines, reactive oxygen species, and extracellular traps (NETs). NETs contain a DNA backbone and catalytically active myeloperoxidase (MPO), which produces hypochlorous acid (HOCl). Chlorination of the DNA nucleoside 8-chloro-deoxyguanosine has been reported as an early marker of inflammation in diabetes. In this study, we examined the reactivity of different chlorinated nucleosides, including 5-chloro-(deoxy)cytidine (5ClC, 5CldC), 8-chloro-(deoxy)adenosine (8ClA, 8CldA) and 8-chloro-(deoxy)guanosine (8ClG, 8CldG), with the INS-1E ß-cell line. Exposure of INS-1E cells to 5CldC, 8CldA, 8ClA, and 8CldG decreased metabolic activity and intracellular ATP, and, together with 8ClG, induced apoptotic cell death. Exposure to 8ClA, but not the other nucleosides, resulted in sustained endoplasmic reticulum stress, activation of the unfolded protein response, and increased expression of thioredoxin-interacting protein (TXNIP) and heme oxygenase 1 (HO-1). Exposure of INS-1E cells to 5CldC also increased TXNIP and NAD(P)H dehydrogenase quinone 1 (NQO1) expression. In addition, a significant increase in the mRNA expression of NQO1 and GPx4 was seen in INS-1E cells exposed to 8ClG and 8CldA, respectively. However, a significant decrease in intracellular thiols was only observed in INS-1E cells exposed to 8ClG and 8CldG. Finally, a significant decrease in the insulin stimulation index was observed in experiments with all the chlorinated nucleosides, except for 8ClA and 8ClG. Together, these results suggest that increased formation of chlorinated nucleosides during inflammation in diabetes could influence ß-cell function and may contribute to disease progression.

Oxygen Exposure and Tolerance Shapes the Cell Wall-Associated Lipids of the Skin Commensal Cutibacterium acnes.

Cutibacterium acnes is one of the most abundant bacteria on the skin. Being exposed to oxygen and oxic stress, the secretion of the bacterial antioxidant protein RoxP ensures an endogenous antioxidant system for the preservation of skin health. To investigate the impact of the antioxidant RoxP on oxidation of the bacteria, wildtype and an isogenic roxp mutant were cultured in anaerobic and oxic conditions. The carbonylated status of proteins were recorded, as were the most significant modifications in a relative intensity of free fatty acids (FFA) and lipids containing fatty acids (FA), such as di- (DG) and triglycerides (TG), di- (DGDG) and sulfoquinozyldiacylglycerol (SQDG) and ceramides. Concerning the fatty acid types, it was observed that the free fatty acids contained mainly C12:0-C26:0 in hydroxy and acylated forms, the DG contained mainly C29:0-C37:0, the TG contained mainly C19:0-C33:0, and the DGDG/SQDGs contained very long fatty acids (C29:0-C37:0) demonstrating the interdependence of de novo synthesis of lipids and RoxP. The area of DGDG peaks (924.52, 929.56 and 930.58) were affected by bacterial growth conditions, with the exception of m/z 910.61. Moreover, the FFA unsaturation is wider in the SQDG species (C30:0 to C36:6) than in DG, TG or free FFA species. It could be concluded that both environmental oxidative statuses, as well as the prevalence of bacterial antioxidant systems, significantly shape the lipidome of C. acnes.

Myeloperoxidase Alters Lung Cancer Cell Function to Benefit Their Survival.

Myeloperoxidase (MPO) is a neutrophil-derived enzyme that has been recently associated with tumour development. However, the mechanisms by which this enzyme exerts its functions remain unclear. In this study, we investigated whether myeloperoxidase can alter the function of A549 human lung cancer cells. We observed that MPO promoted the proliferation of cancer cells and inhibited their apoptosis. Additionally, it increased the phosphorylation of AKT and ERK. MPO was rapidly bound to and internalized by A549 cells, retaining its enzymatic activity. Furthermore, MPO partially translocated into the nucleus and was detected in the chromatin-enriched fraction. Effects of MPO on cancer cell function could be reduced when MPO uptake was blocked with heparin or upon inhibition of the enzymatic activity with the MPO inhibitor 4-aminobenzoic acid hydrazide (4-ABAH). Lastly, we have shown that tumour-bearing mice treated with 4-ABAH had reduced tumour burden when compared to control mice. Our results highlight the role of MPO as a neutrophil-derived enzyme that can alter the function of lung cancer cells.

Effect of Sotagliflozin on Early Mortality and Heart Failure-Related Events: A Post Hoc Analysis of SOLOIST-WHF.

BACKGROUND: Approximately 25% of patients admitted to hospitals for worsening heart failure (WHF) are readmitted within 30 days. OBJECTIVES: The authors conducted a post hoc analysis of the SOLOIST-WHF (Effect of Sotagliflozin on Cardiovascular Events in Patients With Type 2 Diabetes Post-WHF) trial to evaluate the efficacy of sotagliflozin versus placebo to decrease mortality and HF-related events among patients who began study treatment on or before discharge from their index hospitalization. METHODS: The main endpoint of interest was cardiovascular death or HF-related event (HF hospitalization or urgent care visit) occurring within 90 and 30 days after discharge for the index WHF hospitalization. Treatment comparisons were by proportional hazards models, generating HRs, 95% CIs, and P values. RESULTS: Of 1,222 randomized patients, 596 received study drug on or before their date of discharge. Sotagliflozin reduced the main endpoint at 90 days after discharge (HR: 0.54 [95% CI: 0.35-0.82]; P = 0.004) and at 30 days (HR: 0.49 [95% CI: 0.27-0.91]; P = 0.023) and all-cause mortality at 90 days (HR: 0.39 [95% CI: 0.17-0.88]; P = 0.024). In subgroup analyses, sotagliflozin reduced the 90-day main endpoint regardless of sex, age, estimated glomerular filtration rate, N-terminal pro-B-type natriuretic peptide, left ventricular ejection fraction, or mineralocorticoid receptor agonist use. Sotagliflozin was well-tolerated but with slightly higher rates of diarrhea and volume-related events than placebo. CONCLUSIONS: Starting sotagliflozin before discharge in patients with type 2 diabetes hospitalized for WHF significantly decreased cardiovascular deaths and HF events through 30 and 90 days after discharge, emphasizing the importance of beginning sodium glucose cotransporter treatment before discharge.