PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection.

Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve hyper-responsiveness to interferon without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depended on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acted on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to degrade these proteases via the immunoproteasome. Thus, PARP9-DTX3L acted on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

In vitro, in vivo, and in silico analysis of synbiotics as preventive interventions for lipid metabolism in ethanol-induced adipose tissue injury.

The risk of alcoholic liver disease (ALD) is increased by excessive ethanol drinking. For the prevention of ALD, the effects of ethanol on the liver, adipose tissue, and gut are crucial. Interestingly, garlic and a few probiotic strains can protect against ethanol-induced hepatotoxicity. However, the relationship between adipose tissue inflammation, Kyolic aged garlic extract (AGE), and Lactobacillus rhamnosus MTCC1423 in developing ALD is unknown. Therefore, the present study explored the effect of synbiotics (a combination of prebiotics and probiotics) on adipose tissue to prevent ALD. To investigate the efficacy of synbiotics administration on adipose tissue in preventing ALD, in vitro (3T3-L1 cells, N = 3) groups: control, control + LPS (lipopolysaccharide), ethanol, ethanol + LPS, ethanol + synbiotics, ethanol + synbiotics + LPS; in vivo (Wistar male rats, N = 6) groups: control, ethanol, pairfed, ethanol + synbiotics and in silico experiments were conducted. Lactobacillus multiplies in accordance with the growth curve when exposed to AGE. Additionally, Oil red O staining and scanning electron microscopy (SEM) demonstrated that synbiotics therapy maintained the morphology of adipocytes in the alcoholic model. In support of the morphological changes, quantitative real-time PCR demonstrated overexpression of adiponectin and downregulation of leptin, resistin, PPARγ, CYP2E1, iNOS, IL-6, and TNF-α after administration of synbiotics compared to the ethanol group. In addition, MDA estimation by high-performance liquid chromatography (HPLC) indicated that the synbiotics treatment reduced oxidative stress in rat adipose tissue. Consequently, the in-silico analysis revealed that AGE inhibited the C-D-T networks as PPARγ acting as the main target protein. The current study demonstrates that using synbiotics improves adipose tissue metabolism in ALD.

FtlA and FtlB Are Candidates for Inclusion in a Next-Generation Multiantigen Subunit Vaccine for Lyme Disease.

Lyme disease (LD) is a tick-transmitted bacterial infection caused by Borreliella burgdorferi and other closely related species collectively referred to as the LD spirochetes. The LD spirochetes encode an uncharacterized family of proteins originally designated protein family twelve (PF12). In B. burgdorferi strain B31, PF12 consists of four plasmid-carried genes, encoding BBK01, BBG01, BBH37, and BBJ08. Henceforth, we designate the PF12 proteins family twelve lipoprotein (Ftl) A (FtlA) (BBK01), FtlB (BBG01), FtlC (BBH37), and FtlD (BBJ08). The goal of this study was to assess the potential utility of the Ftl proteins in subunit vaccine development. Immunoblot analyses of LD spirochete cell lysates demonstrated that one or more of the Ftl proteins are produced by most LD isolates during cultivation. The Ftl proteins were verified to be membrane associated, and nondenaturing PAGE revealed that FtlA, FtlB, and FtlD formed dimers, while FtlC formed hexamers. Analysis of serum samples from B. burgdorferi antibody (Ab)-positive client-owned dogs (n = 50) and horses (n = 90) revealed that a majority were anti-Ftl Ab positive. Abs to the Ftl proteins were detected in serum samples from laboratory-infected dogs out to 497 days postinfection. Anti-FtlA and FtlB antisera displayed potent complement-dependent Ab-mediated killing activity, and epitope localization revealed that the bactericidal epitopes reside within the N-terminal domain of the Ftl proteins. This study suggests that FtlA and FtlB are potential candidates for inclusion in a multivalent vaccine for LD.

Does calculation method matter for targeting vancomycin area under the curve?

OBJECTIVES: To assess differences in vancomycin AUC estimates from two common, clinically applied first-order pharmacokinetic equation methods compared with Bayesian estimates. METHODS: A cohort of patients who received vancomycin and therapeutic drug monitoring was studied. First-order population pharmacokinetic equations were used to guide initial empirical dosing. After receipt of the first dose, patients had peak and trough serum levels drawn and steady-state AUC was estimated using first-order pharmacokinetic equations as standard care. We subsequently created a Bayesian model and used individual Empirical Bayes Estimates to precisely calculate vancomycin AUC24-48, AUC48-72 and AUC72-96 in this cohort. AUC at steady state (AUCSS) differences from the first-order methods were compared numerically and categorically (i.e. below, within or above 400-600 mg·h/L) to Bayesian AUCs, which served as the gold standard. RESULTS: A total of 65 adult inpatients with 409 plasma samples were included in this analysis. A two-compartment intravenous infusion model with first-order elimination fit the data well. The mean of Bayesian AUC24-48 was not significantly different from AUC estimates from the two first-order pharmacokinetic equation methods (P = 0.68); however, Bayesian AUC48-72 and Bayesian AUC72-96 were both significantly different when compared with both first-order pharmacokinetic equation methods (P < 0.01 for each). At the patient level, categorical classifications of AUC estimates from the two first-order pharmacokinetic equation methods differed from categorizations derived from the Bayesian calculations. Categorical agreement was ∼50% between first-order and Bayesian calculations, with declining categorical agreement observed with longer treatment courses. Differences in categorical agreement between calculation methods could potentially result in different dose recommendations for the patient. CONCLUSIONS: Bayesian-calculated AUCs between 48-72 and 72-96 h intervals were significantly different from first-order pharmacokinetic method-estimated AUCs at steady state. The various calculation methods resulted in different categorical classification, which could potentially lead to erroneous dosing adjustments in approximately half of the patients.

Alterations in bone metabolites with age in C57BL/6 mice model.

Understanding the pathophysiology behind age-related diseases is an urgent need as the elderly population continues to grow. With age, there is a high risk of musculoskeletal deterioration and associated morbidity and mortality. Although the exact mechanism behind age-related degeneration is unknown, it is well established that alteration in cellular metabolism is one of the important contributing factors. Alteration in signaling pathways with age leads to the accumulation or depletion of several metabolites that play a vital role in musculoskeletal pathophysiology. This study aimed to identify age-related changes in bone tissue metabolites in C57BL/6 mice. We then correlated the differentially expressed metabolites with their functions in bone biology. In both aged males and females, hydroxyproline, glutamine, and alpha-linolenic acid levels were decreased. In aged females, Ornithine (p value = 0.001), L-Proline (p value = 0.008), Uridine (p value = 0.001), Aspartic Acid (p value = 0.004) levels were significantly decreased, and glutamate (p value = 0.002) was elevated. In aged males, N-acetyl-D-glucosamine (pvalue = 0.010), Adrenic acid (pvalue = 0.0099), Arachidonic acid (p value = 0.029) and Allantoin (p value = 0.004) levels were decreased. Metabolic pathway analysis revealed that purine and D-glutamine and D-glutamate metabolism were significantly altered in both sexes, while arginine biosynthesis in females and lipid metabolism in males were highly affected. These differences in metabolic signaling might be one of the reasons for the discrepancy in musculoskeletal disease manifestation between the two sexes. Understanding the role of these metabolites play in the aging bone will allow for new sex-specific targeted therapies against the progression of musculoskeletal diseases.

Gut Microbiota: Target for Modulation of Gut-Liver-Adipose Tissue Axis in Ethanol-Induced Liver Disease.

Consumption of alcohol (ethanol) in various forms has been an integral part of human civilization. Since ages, it also has been an important cause of death and health impairment across the globe. Ethanol-mediated liver injury, known as alcoholic liver disease (ALD), is caused by surplus intake of alcohol. Several studies have proposed the different pathways that may be lead to ALD. One of the factors that may affect the cytochrome P450 (CYP2E1) metabolic pathway is gut dysbiosis. The gut microbiota produces various compounds that play an important role in regulating healthy functions of distal organs such as the adipose tissue and liver. Dysbiosis causes bacteremia, hepatic encephalopathy, and increased intestinal permeability. Recent clinical studies have found better understanding of the gut and liver axis. Another factor that may affect the ALD pathway is dysfunction of adipose tissue metabolism. Moreover, dysfunction of adipose tissue leads to ectopic fat deposition within the liver and disturbs lipid metabolism by increasing lipolysis/decreasing lipogenesis and impaired glucose tolerance of adipose tissue which leads to ectopic fat deposition within the liver. Adipokine secretion of resistin, leptin, and adiponectin is adversely modified upon prolonged alcohol consumption. In the combination of these two factors, a proinflammatory state is developed within the patient leading to the progression of ALD. Thus, the therapeutic approach for treatments and prevention for liver cirrhosis patients must be focused on the gut-liver-adipose tissue network modification with the use of probiotics, synbiotics, and prebiotics. This review is aimed at the effect of ethanol on gut and adipose tissue in both rodent and human alcoholic models.

Leachate treatment potential of nanomaterial based assemblies: a systematic review on recent development.

Rapid development of the population has brought about a serious problem of waste generation and management. Open dumping and land filling are two of the preferred options for waste management and treatment. As a consequence of this, the accumulation of leachates has become one of the concerns for environmental sustainability. In this regard, various treatment methodologies have been developed in recent decades. Among them, the nanomaterial-based approaches are the emerging ones in the current scenario due to their various unique properties. Furthermore, nanomaterial-based assemblies (i.e., nanomaterials combined with microbes, chemical catalysts, enzymes, and so on) have been introduced as a novel modification for leachate treatment. This work, therefore, has been dedicated to comprehensively reviewing all nanomaterial based leachate treatment techniques. In this regard, the first part of this review will discuss the nano catalyst, nano adsorbent along with their synthesis and mechanistic view of pollutant removal potential. In the second part, the nanomaterial-based microbial conjugates applied in the leachate treatments have been discussed. Apart from this, various other nanomaterial-based methods have been discussed in the third part of the review. Hence this review is providing an insight of all the recent developments pertaining to the nano material based leachate treatment techniques.

Transformation and Mobility of Cu, Zn, and Cr in Sewage Sludge during Anaerobic Digestion with Pre- or Interstage Hydrothermal Treatment.

Anaerobic digestion (AD) combined with hydrothermal treatment (HT) is an attractive technology for sewage sludge treatment and resource recovery. The fate and distribution of heavy metals in the sludge during combined HT/AD significantly affect the sludge final disposal/utilization options, yet such information is still lacking. This study systematically characterizes the transformation of important heavy metals Cu, Zn, and Cr in sewage sludge during AD with pre- or interstage HT (i.e., HT-AD or AD-HT-AD, respectively). Complementary sequential chemical extraction and X-ray absorption spectroscopy were used to characterize the speciation and mobility of metals. For the HT-AD system, both Cu and Zn predominantly occur as sulfides in HT hydrochars. Subsequent AD favors the formation of Cu2S and partial transformation of nano-ZnS to adsorbed and organo-complexed Zn species. HT favors the formation of Cr-bearing silicates in hydrochars, whereas Fe(III)-Cr(III)-hydroxide and Cr(III)-humic complex are the predominant Cr species in AD solids. Similar reaction pathways occur in the AD-HT-AD system with some minor differences in metal species and contents, as the first-stage AD changed the sludge matrix. These findings have important implications for understanding the fate and mobility of heavy metals in sludge-derived hydrochars and AD solids.

Metformin and Probiotics Interplay in Amelioration of Ethanol-Induced Oxidative Stress and Inflammatory Response in an In Vitro and In Vivo Model of Hepatic Injury.

Alcohol-induced liver injury implicates inflammation and oxidative stress as important mediators. Despite rigorous research, there is still no Food and Drug Administration (FDA) approved therapies for any stage of alcoholic liver disease (ALD). Interestingly, metformin (Met) and several probiotic strains possess the potential of inhibiting alcoholic liver injury. Therefore, we investigated the effectiveness of combination therapy using a mixture of eight strains of lactic acid-producing bacteria, commercialized as Visbiome® (V) and Met in preventing the ethanol-induced hepatic injury using in vitro and in vivo models. Human HepG2 cells and male Wistar rats were exposed to ethanol and simultaneously treated with probiotic V or Met alone as well as in combination. Endoplasmic reticulum (ER) stress markers, inflammatory markers, lipid metabolism, reactive oxygen species (ROS) production, and oxidative stress were evaluated, using qRT-PCR, Oil red O staining, fluorimetry, and HPLC. In vitro, probiotic V and Met in combination prevented ethanol-induced cellular injury, ER stress, oxidative stress, and regulated lipid metabolism as well as inflammatory response in HepG2 cells. Probiotic V and Met also promoted macrophage polarization towards the M2 phenotype in ethanol-exposed RAW 264.7 macrophage cells. In vivo, combined administration of probiotic V and Met ameliorated the histopathological changes, inflammatory response, hepatic markers (liver enzymes), and lipid metabolism induced by ethanol. It also improved the antioxidant markers (HO-1 and Nrf-2), as seen by their protein levels in both HepG2 cells as well as liver tissue using ELISA. Hence, probiotic V may act, in addition to the Met, as an effective preventive treatment against ethanol-induced hepatic injury.

Prophylactic Treatment of Probiotic and Metformin Mitigates Ethanol-Induced Intestinal Barrier Injury: In Vitro, In Vivo, and In Silico Approaches.

Ethanol depletes intestinal integrity and promotes gut dysbiosis. Studies have suggested the individual role of probiotics and metformin Met in protecting intestinal barrier function from injuries induced by ethanol. The objective of the current study is to investigate the potential mechanism by which coadministration of probiotic Visbiome® (V) and Met blocks the ethanol-induced intestinal barrier dysfunction/gut leakiness utilizing Caco-2 monolayers, a rat model with chronic ethanol injury, and in silico docking interaction models. In Caco-2 monolayers, exposure to ethanol significantly disrupted tight junction (TJ) localization, elevated monolayer permeability, and oxidative stress compared with controls. However, cotreatment with probiotic V and Met largely ameliorated the ethanol-induced mucosal barrier dysfunction, TJ disruption, and gut oxidative stress compared with ethanol-exposed monolayers and individual treatment of either agent. Rats fed with ethanol-containing Lieber-DeCarli liquid diet showed decreased expression of TJ proteins, and increased intestinal barrier injury resulting in pro-inflammatory response and oxidative stress in the colon. We found that co-administration of probiotic V and Met improved the expression of intestinal TJ proteins (ZO-1 and occludin) and upregulated the anti-inflammatory response, leading to reduced ER stress. Moreover, co-administration of probiotic V and Met inhibited the CYP2E1 and NOX gene expression, and increase the translocation of Nrf-2 as well as anti-oxidative genes (SOD, catalase, Gpx, and HO-1), leading to reduced colonic ROS content and malondialdehyde levels. The combined treatment of probiotic V and Met also improved their binding affinities towards HO-1, Nrf-2, SLC5A8, and GPR109A, which could be attributed to their synergistic effect. Our findings based on in-vitro, in-vivo, and in-silico analyses suggest that the combination of probiotic V and Met potentially acts in synergism, attributable to their property of inhibition of inflammation and oxidative stress against ethanol-induced intestinal barrier injury.

Protective Effects of Swertiamarin against Methylglyoxal-Induced Epithelial-Mesenchymal Transition by Improving Oxidative Stress in Rat Kidney Epithelial (NRK-52E) Cells.

Increased blood glucose in diabetic individuals results in the formation of advanced glycation end products (AGEs), causing various adverse effects on kidney cells, thereby leading to diabetic nephropathy (DN). In this study, the antiglycative potential of Swertiamarin (SM) isolated from the methanolic extract of E. littorale was explored. The effect of SM on protein glycation was studied by incubating bovine serum albumin with fructose at 60 °C in the presence and absence of different concentrations of swertiamarin for 24 h. For comparative analysis, metformin was also used at similar concentrations as SM. Further, to understand the role of SM in preventing DN, in vitro studies using NRK-52E cells were done by treating cells with methylglyoxal (MG) in the presence and absence of SM. SM showed better antiglycative potential as compared to metformin. In addition, SM could prevent the MG mediated pathogenesis in DN by reducing levels of argpyrimidine, oxidative stress and epithelial mesenchymal transition in kidney cells. SM also downregulated the expression of interleukin-6, tumor necrosis factor-α and interleukin-1ß. This study, for the first time, reports the antiglycative potential of SM and also provides novel insights into the molecular mechanisms by which SM prevents toxicity of MG on rat kidney cells.

Coevolution of Iron, Phosphorus, and Sulfur Speciation during Anaerobic Digestion with Hydrothermal Pretreatment of Sewage Sludge.

Anaerobic digestion (AD) with hydrothermal (HT) pretreatment is an emerging technology for enhanced resource recovery from sewage sludge. This study investigates the speciation of Fe, P, and S during sequential HT-AD treatment of sewage sludge using sequential chemical extraction, X-ray diffraction, and X-ray absorption spectroscopy. Results suggest strong correlations between Fe and P species as well as Fe and S species, affecting the solubility and bioavailability of each other. For instance, much vivianite formed in the hydrochars after HT treatment at low temperature, while more strengite precipitated at higher HT temperature. During the subsequent AD process, microbial reduction of strengite and other Fe(III) species led to the formation of more vivianite, with concurrent P release into the solution and adsorption onto other minerals. HT pretreatment of sewage sludge had a weak effect on the sulfidation of Fe during the AD process. This work has important implications for understanding the nutrient speciation and availability in sludge-derived hydrochars and AD solids. It also provides fundamental knowledge for the selection and optimization of HT pretreatment conditions for enhanced resource recovery through sequential HT-AD process.

Translating the theory of intersectionality into quantitative and mixed methods for empirical gender transformative research on health.

Intersectionality theory has been used primarily in legal research to provide a framework for understanding the experiences of populations situated within multiple systems of oppression, particularly in relation to anti-discrimination law and gender-based violence. Gender transformative approaches to health seek to move beyond individual-level change and instead centre on restructuring the power relationships that create and maintain gender inequalities. Intersectionality theory is well-suited for the study of gender transformation on health, but there is a lack of consensus on clearly defined intersectional methodology in the field of public health, particularly for quantitative studies. Because qualitative methodologies are well-developed and employed with regularity for intersectional health research, the objective of this paper is to describe innovative quantitative and mixed methods approaches underutilised in public health and provide researchers examples of how to design a study's methodology to adequately address intersectional research questions. The proposed methods provide a toolkit for the investigation of complex interactions across multiple levels, which may offer insight into effective points of intervention to reduce disparities, strengthen larger social movements, and ultimately alter structural and policy contexts. Despite challenges posed by the theory, intersectional approaches may be the key to addressing persistent inequalities that limit gender transformation.

Sexually Transmitted Infection Positivity Among Adolescents With or at High-Risk for Human Immunodeficiency Virus Infection in Los Angeles and New Orleans.

BACKGROUND: Gay, bisexual, and transgender youth and homeless youth are at high risk for sexually transmitted infections (STIs). However, little recent data exist describing STI positivity by anatomical site among those groups. We determined the positivity of Chlamydia trachomatis (CT) infection, Neisseria gonorrhoeae (NG) infection, and syphilis antibody reactivity among lesbian, gay, bisexual, transgender, and homeless youth. METHODS: We recruited 1,264 adolescents with high risk behavior aged 12 to 24 years from homeless shelters, lesbian, gay, bisexual, and transgender organizations, community health centers, and using social media and online dating apps in Los Angeles, California and New Orleans, Louisiana from May 2017 to February 2019. Participants received point-of-care pharyngeal, rectal, and urethral/vaginal CT and NG testing and syphilis antibody testing. We calculated STI positivity by anatomical site and compared positivity by participant subgroups based on human immunodeficiency virus (HIV) status, sex assigned at birth, and gender identity. RESULTS: CT and NG positivity and syphilis antibody reactivity was higher among HIV-infected adolescent men who have sex with men (MSM) than HIV-uninfected adolescent MSM (40.2% vs. 19%, P < 0.05), particularly CT or NG rectal infection (28% vs. 12.3%, P < 0.05). Of participants with positive CT or NG infections, 65% had extragenital-only infections, 20% had both extragenital and urogenital infections, and 15% had urogenital-only infections. CONCLUSIONS: Sexually transmitted infection positivity was high, particularly among transgender women and MSM. The high proportion of rectal and pharyngeal infections highlights the importance of both urogenital and extragenital STI screening. More accessible STI testing is necessary for high-risk adolescent populations.

First comprehensive structural and biophysical analysis of MAPK13 inhibitors targeting DFG-in and DFG-out binding modes.

BACKGROUND: P38 MAP kinases are centrally involved in mediating extracellular signaling in various diseases. While much attention has previously been focused on the ubiquitously expressed family member MAPK14 (p38α), recent studies indicate that family members such as MAPK13 (p38δ) display a more selective cellular and tissue expression and might therefore represent a specific kinase to target in certain diseases. METHODS: To facilitate the design of potent and specific inhibitors, we present here the structural, biophysical, and functional characterization of two new MAPK13-inhibitor complexes, as well as the first comprehensive structural, biophysical, and functional analysis of MAPK13 complexes with four different inhibitor compounds of greatly varying potency. RESULTS: These inhibitors display IC50 values either in the nanomolar range or micromolar range (>800-fold range). The nanomolar inhibitors exhibit much longer ligand-enzyme complex half-lives compared to the micromolar inhibitors as measured by biolayer interferometry. Crystal structures of the MAPK13 inhibitor complexes reveal that the nanomolar inhibitors engage MAPK13 in the DFG-out binding mode, while the micromolar inhibitors are in the DFG-in mode. Detailed structural and computational docking analyses suggest that this difference in binding mode engagement is driven by conformational restraints imposed by the chemical structure of the inhibitors, and may be fortified by an additional hydrogen bond to MAPK13 in the nanomolar inhibitors. CONCLUSIONS: These studies provide a structural basis for understanding the differences in potency exhibited by these inhibitors. GENERAL SIGNIFICANCE: They also provide the groundwork for future studies to improve specificity, potency, pharmacodynamics, and pharmacokinetic properties.

Protective Effects of Diallyl Sulfide Against Ethanol-Induced Injury in Rat Adipose Tissue and Primary Human Adipocytes.

BACKGROUND: Alcohol consumption is the fourth leading cause of death and disability worldwide. Several cellular pathways contribute to alcohol-mediated tissue injury. Adipose tissue apart from functioning as an endocrine organ secretes several hormones and cytokines known as adipokines that are known to play a significant role in alcohol-induced tissue damage. This study was designed to test the efficacy of diallyl sulfide (DAS) in regulating the alcohol-induced outcomes on adipose tissue. METHODS: Male Wistar rats were fed with 36% Lieber-DeCarli liquid diet containing ethanol (EtOH) for 4 weeks. Control rats were pair-fed with isocaloric diet containing maltodextrin instead of EtOH. During the last week of feeding protocol, the EtOH-fed rat group was given 200 mg/kg body weight of DAS through diet. We also studied DAS effect on isolated human primary adipocytes. Viability of human primary adipocytes on DAS treatment was assessed by MTT assay. Malondialdehyde (MDA), a marker of oxidative stress, was measured by HPLC and the thiobarbituric acid method. Expression of inflammatory genes and lipogenic genes was studied by qRT-PCR and Western blotting. Serum inflammatory gene expression was studied by ELISA. RESULTS: Our study results showed that DAS could alleviate EtOH-induced expression levels of proinflammatory and endoplasmic reticulum (ER) stress genes and improve adipose tissue mass and adipocyte morphology in male Wistar rats fed Lieber-DeCarli diet containing 6% EtOH. Further, we showed that DAS reduced the expression of lipogenic genes and improved lipid accumulation and adipocyte mass in human primary adipocytes treated with EtOH. Subsequently, we also showed that oxidative stress, as measured by the changes in MDA levels, was reduced in both male Wistar rats and human primary adipocytes treated with EtOH plus DAS. CONCLUSIONS: Our study results prove that DAS is effective in ameliorating EtOH-induced damage to adipose tissue as evidenced by the reduction brought about by DAS in oxidative stress, ER stress, and proinflammatory gene expression levels. DAS treatment also regulated lipogenic gene expression levels, thereby reducing free fatty acid release. In conclusion, this study has clinical implications with respect to alcohol-induced adipose tissue injury among alcohol users.

Redox Mechanisms Influencing cGMP Signaling in Pulmonary Vascular Physiology and Pathophysiology.

The soluble form of guanylate cyclase (sGC) and cGMP signaling are major regulators of pulmonary vasodilation and vascular remodeling that protect the pulmonary circulation from hypertension development. Nitric oxide, reactive oxygen species, thiol and heme redox, and heme biosynthesis control mechanisms regulating the production of cGMP by sGC. In addition, a cGMP-independent mechanism regulates protein kinase G through thiol oxidation in manner controlled by peroxide metabolism and NADPH redox. Multiple aspects of these regulatory processes contribute to physiological and pathophysiological regulation of the pulmonary circulation, and create potentially novel therapeutic targets for the treatment of pulmonary vascular disease.

Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells.

Voltage-gated L-type Ca(2+) current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2 We studied whether superoxide per se acutely modulates ICa,L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 µM rotenone. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 µM) resulted in a greater ICa,L increase in a dose-dependent manner to a maximum of 22.1% at 10 µM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced ICa,L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in ICa,L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger ICa,L preceding its dismutation to H2O2 The removal of nitric oxide is a likely mechanism for the increase in ICa,L.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition.

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.