Alterations in genes associated with cytosolic RNA sensing in whole blood are associated with coronary microvascular disease in SLE.

Systemic lupus erythematosus (SLE) patients are 90% women and over three times more likely to die of cardiovascular disease than women in the general population. Chest pain with no obstructive cardiac disease is associated with coronary microvascular disease (CMD), where narrowing of the small blood vessels can lead to ischemia, and frequently reported by SLE patients. Using whole blood RNA samples, we asked whether gene signatures discriminate SLE patients with coronary microvascular dysfunction (CMD) on cardiac MRI (n=4) from those without (n=7) and whether any signaling pathway is linked to the underlying pathobiology of SLE CMD. RNA-seq analysis revealed 143 differentially expressed (DE) genes between the SLE and healthy control (HC) groups, with virus defense and interferon (IFN) signaling being the key pathways identified as enriched in SLE as expected. We next conducted a comparative analysis of genes differentially expressed in SLE-CMD and SLE-non-CMD relative to HC samples. Our analysis highlighted differences in IFN signaling, RNA sensing and ADP-ribosylation pathways between SLE-CMD and SLE-non-CMD. This is the first study to investigate possible gene signatures associating with CMD in SLE, and our data strongly suggests that distinct molecular mechanisms underly vascular changes in CMD and non-CMD involvement in SLE.

α-Ketoglutarate-Dependent KDM6 Histone Demethylases and Interferon-Stimulated Gene Expression in Lupus.

OBJECTIVE: We aimed to investigate the hypothesis that interferon (IFN)-stimulated gene (ISG) expression in systemic lupus erythematosus (SLE) monocytes is linked to changes in metabolic reprogramming and epigenetic regulation of ISG expression. METHODS: Monocytes from healthy volunteers and patients with SLE at baseline or following IFNα treatment were analyzed by extracellular flux analysis, proteomics, metabolomics, chromatin immunoprecipitation, and gene expression. The histone demethylases KDM6A/B were inhibited using glycogen synthase kinase J4 (GSK-J4). GSK-J4 was tested in pristane and resiquimod (R848) models of IFN-driven SLE. RESULTS: SLE monocytes had enhanced rates of glycolysis and oxidative phosphorylation compared to healthy control monocytes, as well as increased levels of isocitrate dehydrogenase and its product, α-ketoglutarate (α-KG). Because α-KG is a required cofactor for histone demethylases KDM6A and KDM6B, we hypothesized that IFNα may be driving "trained immune" responses through altering histone methylation. IFNα priming (day 1) resulted in a sustained increase in the expression of ISGs in primed cells (day 5) and enhanced expression on restimulation with IFNα. Importantly, decreased H3K27 trimethylation was observed at the promoters of ISGs following IFNα priming. Finally, GSK-J4 (KDM6A/B inhibitor) resulted in decreased ISG expression in SLE patient monocytes, as well as reduced autoantibody production, ISG expression, and kidney pathology in R848-treated BALB/c mice. CONCLUSION: Our study suggests long-term IFNα exposure alters the epigenetic regulation of ISG expression in SLE monocytes via changes in immunometabolism, a mechanism reflecting trained immunity to type I IFN. Importantly, it opens the possibility that targeting histone-modifying enzymes, such as KDM6A/B, may reduce IFN responses in SLE.

Reduced left ventricular function on cardiac MRI of SLE patients correlates with measures of disease activity and inflammation.

Background: Women with SLE have an elevated risk of cardiovascular disease. Many women with SLE frequently report chest pain in the absence of obstructive coronary artery disease (CAD) due to coronary microvascular dysfunction (CMD), a form of ischemia with no obstructive CAD. Echocardiographic studies have shown that SLE patients have reduced left ventricular (LV) function, which may also correlate with higher SLE disease activity scores. As such, we used cardiac magnetic resonance imaging (cMRI) to investigate the relationship between SLE, related inflammatory biomarkers, and cardiac function in female SLE patients. Methods: We performed stress cMRI in women with SLE and chest pain with no obstructive CAD (n=13, all met ACR 1997 criteria,) and reference controls (n=22) using our published protocol. We evaluated LV function, tissue characterization (T1 mapping, ECV), and delayed enhancement, using CV142 software (Circle Cardiovascular Imaging Inc, Calgary, AB, Canada). Myocardial perfusion reserve index (MPRI) was calculated using our published protocol. SLEDAI and SLICC Damage Index (DI) were calculated per validated criteria. Serum samples were analyzed for inflammatory markers and autoantibodies. Wilcoxon rank-sum test was performed on clinical values with CMD and no CMD SLE subjects, and on cMRI values with all SLE subjects and controls. Correlation analysis was done on clinical values, and cMRI values on all SLE subjects. Results: Overall, 40% of SLE subjects had MPRI values < 1.84, consistent with CMD. Compared to controls, SLE subjects had significantly lower LVEF, and higher LVESVi and LVMi. Corresponding to this, radial, longitudinal, and circumferential strain were significantly lower in the SLE subjects. In correlation analysis of serum inflammatory biomarkers to cMRI values in the SLE subjects, SLICC DI was related to worse cardiac function (lower radial, circumferential and longitudinal strain) and higher T1 time. Additionally, fasting insulin and ESR were negatively correlated with LVMi. Fasting insulin also negatively correlated with ECV. CRP had a positive association with LVESV index and CI and a negative association with longitudinal strain. Conclusions: Among women with SLE with chest pain and no obstructive CAD, 40% have CMD. While evaluations of known inflammatory markers (such as CRP and ESR) predictably correlated with decreased cardiac function, our study found that decreased fasting insulin levels as a novel marker of diminished LV function. In addition, low insulin levels were observed to correlate with increased LVMi and ECV, suggesting a cardioprotective effect of insulin in SLE patients. We also noted that SLICC DI, an assessment of SLE damage, correlates with cardiac dysfunction in SLE. Our findings underline the potential of non-invasive cMRI as a tool for monitoring cardiovascular function in SLE, particularly in patients with high SLICC DI, ESR and CRP and low fasting insulin levels.

Oxidative stress-A key determinant of complications and negative outcome in hepatitis E virus infected pregnancies: A comprehensive account involving cases from northeast India.

Regulated oxidative stress (OS) is important during pregnancy. Sporadic studies suggest the significance of deregulated OS in hepatitis E virus (HEV) infected pregnancy, but with limited reactive oxygen species (ROS) or antioxidant markers. The present novel study, therefore, aimed to evaluate the significance of ROS-antioxidant imbalance and resulting altered OS in HEV infected pregnancy complications like preterm delivery (PTD) and outcome. Difference in serum levels of ROS and antioxidant panel of markers were evaluated by ELISA for HEV immunoglobulin M RNA positive genotype 1 cases (including acute [acute viral hepatitis, AVH] and fulminant [fulminant hepatic failure, FHF] cases) and healthy term delivery subjects, and analyzed statistically. Direct ROS marker H2 O2 levels and indirect OS marker for DNA damage 8-hydroxy-2'-deoxyguanosine was significantly increased in HEV-cases compared to controls, and was associated and prognostic factor for PTD and fetal death in HEV cases. A comparatively lower total serum antioxidant capacity was observed in the FHF cases compared to the control subjects and the AVH cases. Glutathione (GSH) levels and superoxide dismutase (SOD) activity were significantly associated with PTD in the FHF sub-cohorts (p = 0.017) and AVH sub-cohorts (p < 0.001), respectively, and was associated with poor prognosis in HEV cases. The serum H2 O2 levels were found to be negatively correlated with SOD activity (p = 0.016) and GSH levels (p = 0.001) in the HEV-AVH cases; and positively correlated with the viral load in HEV cases (p = 0.023). The ROS-antioxidant imbalance resulting OS plays a detrimental associative role in HEV infected pregnancy complications like PTD and adverse pregnancy outcomes; and holds therapeutic significance.

Reduced Left Ventricular Function on Cardiac MRI in SLE Patients Correlates with Measures of SLE Disease Activity and Inflammation.

Background: Women with SLE have an elevated risk of CVD morbidity and mortality and frequently report chest pain in the absence of obstructive CAD. Echocardiographic studies often demonstrate reduced LV function, correlating with higher disease activity. We used cardiac MRI (cMRI) to investigate the relationship between SLE, related inflammatory biomarkers and cardiac function in female SLE patients. Methods: Women with SLE reporting chest pain with no obstructive CAD (n=13) and reference controls (n=22) were evaluated using stress-rest cMRI to measure LV structure, function, tissue characteristics, and myocardial perfusion reserve index (MPRI). Coronary microvascular dysfunction (CMD) was defined as MPRI <1.84. Serum samples were analyzed for inflammatory markers. Relationships between clinical and cMRI values of SLE subjects were assessed, and groups were compared. Results: 40% of SLE subjects had MPRI < 1.84 on cMRI. Compared to controls, SLE subjects had higher LV volumes and mass and lower LV systolic function. SLICC DI was related to worse cardiac function and higher T1. CRP was related to higher cardiac output and a trend to better systolic function, while ESR and fasting insulin were related to lower LV mass. Lower fasting insulin levels correlated with increased ECV. Conclusions: Among our female SLE cohort, 40% had CMD, and SLICC DI correlated with worse cardiac function and diffuse fibrosis. Higher inflammatory markers and low insulin levels may associate with LV dysfunction. Our findings underline the potential of non-invasive cMRI as a tool for monitoring cardiovascular function in SLE patients.

Sex bias in systemic lupus erythematosus: a molecular insight.

Acknowledging sex differences in immune response is particularly important when we consider the differences between men and women in the incidence of disease. For example, over 80% of autoimmune disease occurs in women, whereas men have a higher incidence of solid tumors compared to women. In general women have stronger innate and adaptive immune responses than men, explaining their ability to clear viral and bacterial infections faster, but also contributing to their increased susceptibility to autoimmune disease. The autoimmune disease systemic lupus erythematosus (SLE) is the archetypical sexually dimorphic disease, with 90% of patients being women. Various mechanisms have been suggested to account for the female prevalence of SLE, including sex hormones, X-linked genes, and epigenetic regulation of gene expression. Here, we will discuss how these mechanisms contribute to pathobiology of SLE and how type I interferons work with them to augment sex specific disease pathogenesis in SLE.

Oxygen Activation by a Copper Complex with Sulfur-Only Coordination Relevant to the Formylglycine Generating Enzyme.

Synthesizing hydrosulfido Cu thiolate complexes is quite challenging. In this report, two new and rare hydrosulfido Cu thiolate complexes, [Et4N]2[(mnt)Cu-SH] (2, mnt = maleonitrile dithiolene = S2C2(CN)2) and [Et4N]3[(mnt)Cu-(µ-SH)-Cu(mnt)] (3), have been synthesized. Coordination sites and O2 activation by complex 2 resemble the formylglycine generating enzyme (FGE), an enzyme recently crystallographically characterized with sulfur-only coordination around Cu (three thiolate ligands). The function of this enzyme (and complex 2) is surprising because vulnerable thiolates should not be well suited for O2 activation rationally. Indeed, activation of oxygen by such an all-sulfur-coordinated Cu complex 2 is lacking in the literature. Aerial O2 (ambient O2 from the air) activation by complex 2 could proceed through a superoxide radical intermediate and a sulfur radical intermediate detected by resonance Raman (rR) spectroscopy and electron paramagnetic resonance (EPR) spectroscopy, respectively. The chemistry of 2 has been examined by its reactivity, crystal structure, and spectroscopic and cyclic voltammetric analyses. In addition, the results have been complemented with density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations.

Prognostic, clinical, and therapeutic importance of RANTES-CCR5 axis in hepatitis A infection: A multiapproach study.

Fulminant hepatic failure (FHF) is a lethal manifestation of hepatitis A virus (HAV) infection, whose underlying mechanisms are poorly understood. We aimed to evaluate the importance of the modulation of the RANTES-chemokine receptor type 5 (CCR5) signaling axis and its immunomodulatory effects in directing hepatitis A disease pathogenesis using an in silico, in vitro and patient cohort-based approach. In silico interaction studies were performed using computation approaches with suitable software. Differential expression of relevant cytokines and immune cell markers were studied using real-time quantitative reverse transcription PCR (qRT-PCR), enzyme-linked immunosorbent assay, and flow-cytometry-based methods. In the HepG2 cell line, we studied inflammatory responses and susceptibility to HAV infection following RANTES stimulation and antibody blockade of CCR5. The HAV-VP3 region exhibited high interaction in CCR5: HAV complexes. RANTES levels were significantly increased in FHF cases. Reduced monocyte and T-cell activation were observed in FHF cases. RANTES expression inversely correlated with viremia but positively correlated with proinflammatory responses. Hyper Th1-biased immune responses, marked by high interleukin (IL)-12/IL-10 ratio were observed in FHF cases, which were also characterized by upregulated tumor necrosis factor-alpha (TNF-α) expression and reduced interferon-gamma expression. In vitro, RANTES was protective against HAV infection but resulted in upregulated TNF-α expression. Although viral load increased upon the regulation of inflammatory responses by CCR5 blocking, it was still significantly lower compared to control HAV-infected cells. Our study suggests the importance of RANTES-CCR5 signaling and linked-immunomodulation in HAV disease pathogenesis, as well as highlights the utility of CCR5 antagonists as a risk-reduction strategy in FHF patients. Our findings, therefore, have important implications for the management of high-risk HAV infections.

A Dithiolato and Hydrido Bridged (CO/CN)Fe-Ni Complex with Unprotected CN: A Model for the [Ni-R] State of the [Ni-Fe] Hydrogenase Active Site.

A dithiolate/hydride bridged Fe-Ni complex, [(CN)(CO)2FeII(µ-pdt)(µ-H)NiII(CN)(PCy3)]- (2, pdt = propane-1,3-dithiolate) has been synthesized by the reaction of [(CN)2(CO)2FeII(pdt)]2- with [NiII(Cl)(H)(PCy3)2] as a synthetic analogue of the Ni-R state of the active site of the [Ni-Fe] hydrogenase. X-ray crystallography of this model complex suggests that the hydride unsymmetrically binds to Ni and Fe similar to natural [Ni-Fe] hydrogenases.

Graphene Supported Rhodium Nanoparticles for Enhanced Electrocatalytic Hydrogen Evolution Reaction.

Current research on catalysts for proton exchange membrane fuel cells (PEMFC) is based on obtaining higher catalytic activity than platinum particle catalysts on porous carbon. In search of a more sustainable catalyst other than platinum for the catalytic conversion of water to hydrogen gas, a series of nanoparticles of transition metals viz., Rh, Co, Fe, Pt and their composites with functionalized graphene such as RhNPs@f-graphene, CoNPs@f-graphene, PtNPs@f-graphene were synthesized and characterized by SEM and TEM techniques. The SEM analysis indicates that the texture of RhNPs@f-graphene resemble the dispersion of water droplets on lotus leaf. TEM analysis indicates that RhNPs of <10 nm diameter are dispersed on the surface of f-graphene. The air-stable NPs and nanocomposites were used as electrocatalyts for conversion of acidic water to hydrogen gas. The composite RhNPs@f-graphene catalyses hydrogen gas evolution from water containing p-toluene sulphonic acid (p-TsOH) at an onset reduction potential, Ep, -0.117 V which is less than that of PtNPs@f-graphene (Ep, -0.380 V) under identical experimental conditions whereas the onset potential of CoNPs@f-graphene was at Ep, -0.97 V and the FeNPs@f-graphene displayed onset potential at Ep, -1.58 V. The pure rhodium nanoparticles, RhNPs also electrocatalyse at Ep, -0.186 V compared with that of PtNPs at Ep, -0.36 V and that of CoNPs at Ep, -0.98 V. The electrocatalytic experiments also indicate that the RhNPs and RhNPs@f-graphene are stable, durable and they can be recycled in several catalytic experiments after washing with water and drying. The results indicate that RhNPs and RhNPs@f-graphene are better nanoelectrocatalysts than PtNPs and the reduction potentials were much higher in other transition metal nanoparticles. The mechanism could involve a hydridic species, Rh-H- followed by interaction with protons to form hydrogen gas.

Preparation of Iron-Sulfur Cubane Nanoclusters and Their Immobilization on Functionalized Carbon Nanotubes.

With large surface area and versatile electronic behaviour, the composites of Fe4S4(SRS)4 nanoclusters and functionalized carbon nanotubes (f-CNTs), are expected to catalyze the conversion of protons to hydrogen gas at lower electro-potentials with higher output than a platinum electrode. In search of a non-noble metal based catalytic material, we report for the first time, the isolation of unimolecular iron-sulfur cubane cluster, [Fe4(µ-S)4(mnt)4], (1) (mnt = maleonitriledithiolate) as nanocubes (63×85×120 nm) in MeCN-EtOH (MeCN is acetonitrile, while EtOH is ethanol) solvents and bimolecular [NBu4]4[Fe4(µ-S)4(mnt)4] as nanocuboctahedra (120×121×125 nm) in pure EtOH. The cubic shape of the nanocrystal reminds its geometrical relationship with a molecular cube and one of sides of the nanocube and nanocuboctahedron matches at 120 nm. The nanocubes of Fe4S4(SRS)4 have been immobilized on f-CNTs and characterized by SEM and TEM methods which indicate that clusters of Fe4S4 of diameter (8-9 nm) interact with surfaces, sidewalls and tip of the f-CNTs. A ferrocene type of interaction could not be observed with f-CNTs, because nanoparticles are not found in CNT-inner cavity. The interaction could be either adsorption or hydrogen bonding interactions between -COOH/-OH groups of f-CNTs and N≡C terminals of iron-sulphur nanoclusters leading to immobilization of an iron-sulfur nanocluster on a single CNT molecule or the iron-sulfur nanoclusters can be entrapped between two CNT molecules.

Multidrug-resistant Pseudomonas aeruginosa from sputum of patients with cystic fibrosis demonstrates a high rate of susceptibility to ceftazidime-avibactam.

PURPOSE: Ceftazidime-avibactam is a novel antimicrobial combining a third-generation cephalosporin with a non-ß-lactam ß-lactamase inhibitor that was recently approved to treat Gram-negative hospital- and ventilator-acquired pneumonia. The use of ceftazidime-avibactam to treat Pseudomonas aeruginosa respiratory infections in patients with cystic fibrosis (CF) has not been evaluated. In this study, we assessed the ceftazidime-avibactam susceptibility of multidrug-resistant (MDR) P. aeruginosa sputum isolates from adults with CF. METHODS: Sputum was collected from individuals with CF, aged ≥18 years, known to be colonized with MDR P. aeruginosa, and tested for susceptibility to 11 different antipseudomonal antimicrobial agents. Isolates were included in the analysis if they were resistant to both ceftazidime and at least one agent in ≥3 different antimicrobial categories routinely used to treat P. aeruginosa. Subject demographics and clinical characteristics were collected. Ceftazidime-avibactam-resistant isolates were screened for the presence of ß-lactam-resistant mechanisms. RESULTS: Thirty-two P. aeruginosa isolates were analyzed, of which 23 isolates were sensitive to ceftazidime-avibactam (71.9%). Ten of the isolates were mucoid and 22 isolates were nonmucoid, both demonstrating >70% susceptibility to ceftazidime-avibactam. The most notable difference in the subjects with resistant strains was an older age and lower body mass index (BMI). Ceftazidime-avibactam-resistant strains showed elevated AmpC expression in >60% of the strains and loss of OprD detection in >70% of the strains. CONCLUSION: Ceftazidime-avibactam demonstrated a significant in vitro activity against highly resistant P. aeruginosa sputum isolates from individuals with CF. Further evaluation of the cause of resistance and clinical impact of ceftazidime-avibactam in CF patients with MDR P. aeruginosa is warranted.

Cigarette Smoke Toxins-Induced Mitochondrial Dysfunction and Pancreatitis Involves Aryl Hydrocarbon Receptor Mediated Cyp1 Gene Expression: Protective Effects of Resveratrol.

We previously reported that mitochondrial CYP1 enzymes participate in the metabolism of polycyclic aromatic hydrocarbons and other carcinogens leading to mitochondrial dysfunction. In this study, using Cyp1b1-/-, Cyp1a1/1a2-/-, and Cyp1a1/1a2/1b1-/- mice, we observed that cigarette and environmental toxins, namely benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induce pancreatic mitochondrial respiratory dysfunction and pancreatitis. Our results suggest that aryl hydrocarbon receptor (AhR) activation and resultant mitochondrial dysfunction are associated with pancreatic pathology. BaP treatment markedly inhibits pancreatic mitochondrial oxygen consumption rate (OCR), ADP-dependent OCR, and also maximal respiration, in wild-type mice but not in Cyp1a1/1a2-/- and Cyp1a1/1a2/1b1-/- mice. In addition, both BaP and TCDD treatment markedly affected mitochondrial complex IV activity, in addition to causing marked reduction in mitochondrial DNA content. Interestingly, the AhR antagonist resveratrol, attenuated BaP-induced mitochondrial respiratory defects in the pancreas, and reversed pancreatitis, both histologically and biochemically in wild-type mice. These results reveal a novel role for AhR- and AhR-regulated CYP1 enzymes in eliciting mitochondrial dysfunction and cigarette toxin-mediated pancreatic pathology. We propose that increased mitochondrial respiratory dysfunction and oxidative stress are involved in polycyclic aromatic hydrocarbon associated pancreatitis. Resveratrol, a chemo preventive agent and AhR antagonist, and CH-223191, a potent and specific AhR inhibitor, confer protection against BaP-induced mitochondrial dysfunction and pancreatic pathology.

17ß-Estradiol Dysregulates Innate Immune Responses to Pseudomonas aeruginosa Respiratory Infection and Is Modulated by Estrogen Receptor Antagonism.

Females have a more severe clinical course than males in terms of several inflammatory lung conditions. Notably, females with cystic fibrosis (CF) suffer worse outcomes, particularly in the setting of Pseudomonas aeruginosa infection. Sex hormones have been implicated in experimental and clinical studies; however, immune mechanisms responsible for this sex-based disparity are unknown and the specific sex hormone target for therapeutic manipulation has not been identified. The objective of this study was to assess mechanisms behind the impact of female sex hormones on host immune responses to P. aeruginosa We used wild-type and CF mice, which we hormone manipulated, inoculated with P. aeruginosa, and then examined for outcomes and inflammatory responses. Neutrophils isolated from mice and human subjects were tested for responses to P. aeruginosa We found that female mice inoculated with P. aeruginosa died earlier and showed slower bacterial clearance than males (P < 0.0001). Ovariectomized females supplemented with 17ß-estradiol succumbed to P. aeruginosa challenge earlier than progesterone- or vehicle-supplemented mice (P = 0.0003). 17ß-Estradiol-treated ovariectomized female mice demonstrated increased lung levels of inflammatory cytokines, and when rendered neutropenic the mortality difference was abrogated. Neutrophils treated with 17ß-estradiol demonstrated an enhanced oxidative burst but decreased P. aeruginosa killing and earlier cell necrosis. The estrogen receptor (ER) antagonist ICI 182,780 improved survival in female mice infected with P. aeruginosa and restored neutrophil function. We concluded that ER antagonism rescues estrogen-mediated neutrophil dysfunction and improves survival in response to P. aeruginosa ER-mediated processes may explain the sex-based mortality gap in CF and other inflammatory lung illnesses, and the ER blockade represents a rational therapeutic strategy.

Molecular epidemiology of HBV infection in chronic hepatitis B virus infected patients in northeast India.

The present study aimed to evaluate the molecular epidemiology of HBV in chronic HBV infected cases from northeast India (NEI), since scanty data are available from the region which has a predominant ethnically distinct tribal population. A total of 523 clinically diagnosed index chronic HBV infected cases and 172 asymptomatic patients (based on family screening) were enrolled with informed consent. Patients were stratified based on serology, imaging, pathology, and clinical data and grouped as chronic HBV and cirrhotic cohorts. Analysis for serum HBV DNA levels and HBV genotyping was performed, and was statistically co-related with disease severity. Males were more prone to chronic HBV infection. Majority of the patients who had Chronic HBV infection based on family screening were females (59.88%), majorly wives of index patients. Mean viral load in Chronic HBV patients was almost 4.5-folds higher than cirrhosis patients, and was significantly associated with e-antigen positive status(P < 0.001) in both groups. HBV genotype D was the most prevalent genotype (62.30%) in NEI. Mixed genotype infection of A + D was found from Assam, along with C + D cases (1.29%) cumulatively. There is a high prevalence of HBV genotype C (13.96%) in our studied cohort which was found to be associated with higher viral load(P = 0.018), e-antigen positivity(P = 0.043), and increased cirrhosis risk compared to Chronic HBV cases [OR = 1.670]. Family contacts in NEI are prone to infection with HBV and development of Chronic HBV. Higher presence of e-positive cases and genotype C along with the mixed genotypes in NEI is unique and of significance with reference to predisposition to disease severity and even response to antiviral therapy.

Molecular epidemiology of hepatitis A virus infection in Northeast India.

The present study was undertaken to screen the molecular epidemiology of Hepatitis A virus (HAV) in Northeast India (NEI) who are ethnically distinct, tribal dominated and of lower socio-economic status with almost no information available from NEI on these aspects. Briefly, 3 ml blood was collected from 324 random liver disease cases with jaundice, receiving care at Central Hospital, N.F. Railway, Guwahati, Assam with informed consent. The patients detected with HAV-IgM positive status were included and were stratified as acute viral hepatitis (AVH) and fulminant hepatitis (FHF) based on clinical profile. Viral RNA was isolated and HAV-RNA was detected by Real-time PCR using primers for the VP3-VP1 region. HAV genotyping was studied by PCR-direct sequencing-phylogenetic analysis approach using the VP1/2A region of HAV isolates. Statistical analysis was performed using SPSS13.0 software. A total of 69 cases were HAV infected with two HBV co-infected cases (n = 69 + 2 = 71), 62 cases and two co-infected cases were AVH and others were FHF cases. HAV infection was predominant in especially in the young and adult age group. HAV-RNA was detected in 28 cases, out of which 19 cases could be genotyped (12 AVH, 7 FHF); which showed the prevalence of genotype IIIA or IA only. Although HAV genotype IIIA was the major genotype in both the AVH (10/12, 83.33%) and FHF (5/7, 71.43%) group, but the difference in distribution of genotypes in AVH and FHF cases was statistically non-significant (P = 0.550). HAV genotype IIIA is associated with the majority of HAV infected cases and severity in NEI.

The ferrous-dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: the role of proximal histidine on its stability.

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O2 dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790-16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O2 are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25°C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O2 at a rate of ~10s(-1). To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate=6.1s(-1)). Decomposition of the oxygenated intermediate is independent of O2 concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.

A cyanide-bridged molybdenum bis(maleonitriledithiolate) square.

[Et4N]2[Mo(IV)O(mnt)2] (mnt = maleonitriledithiolate) reacts, as a synthon, with Me3SiCN under an acidic medium to produce the square complex [Et4N]4[Mo4(µ-CN)4(mnt)8] (1) in high yield. Complex 1 shows strong antiferromagnetic interactions between adjacent Mo atoms in the cluster. The presence of redox-active mnt as a capping ligand strongly influences the magnetic property of 1. The physicochemical properties of 1 have been rationalized by density functional theory level of calculations.

Identification of proximal and distal axial ligands in Leishmania major pseudoperoxidase.

Previous optical and electron paramagnetic resonance (EPR) spectroscopic studies of the newly discovered peroxynitrite scavenging pseudoperoxidase from Leishmania major (LmPP) suggested that ferric LmPP contained a six-coordinate low-spin (6cLS) heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. To identify the axial ligands of LmPP, we exploit a systematic mutational analysis of potential heme ligands. On the basis of UV-visible and EPR spectroscopy, we report that the substitution of the proximal His206 with alanine in LmPP alters the 6cLS to a five-coordinate high spin (5cHS) form at pH 4.0 that has a spectrum characteristic of a Cys-ligated 5cHS derivative. The electronic absorption and EPR analysis of all alanine-substituted Cys and Met single mutants establish that when Cys107 is replaced with alanine, a new species appears that has a spectrum characteristic of a histidine-ligated 5cHS derivative at pH 4.0. Together, these results suggest that His206 and Cys107 act as the proximal and distal axial ligands in ferric LmPP, respectively. However, the electronic properties of reduced wild-type LmPP are similar to those of known 5cHS His-ligated heme proteins at pH 8.8, indicating that the thiolate bond was broken upon reduction. Furthermore, the wild-type protein was only partially reduced at pH 4.0, but the E105L mutant was completely reduced to form a 5cHS ferrous heme. These results imply that the presence of an acidic residue near the distal site may prevent reduction of the heme iron at acidic pH.

Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O2-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O2-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O2 tensions.