DNA polymerase ß of Leishmania donovani is important for infectivity and it protects the parasite against oxidative damage.

The visceral leishmaniasis is caused by L. donovani, a neglected tropical disease with an estimated number of 500,000 cases worldwide. Apart from the absence of effective vaccine, the available drugs have limitations like toxic side effects and emergence of drug resistance. The genome of Leishmania is remarkably challenged by the oxidative stress present inside the human macrophage. To maintain genomic integrity, a number of specialized DNA repair pathways assist in the recognition and repair of damaged DNA. In general, Base Excision Repair (BER) plays an essential role in the maintenance of genomic stability. We demonstrate here that the treatment of L. donovani with oxidative agents causes DNA damage and upregulation of Polß. On the other hand, parasite overexpressing Polß shows more resistance against Amp B, H2O2 and menadione as compared to wild type cells. We also observed a higher infectivity in the parasites that overexpress Polß. The upregulation of Polß was also found in stationary phase and axenic amastigote of L. donovani. Overall, we propose that Polß is crucial for infectivity and survival of the parasite. Discovery of specific inhibitors against Polß could offer an attractive strategy against leishmaniasis.

Oxidative Stress-Mediated Overexpression of Uracil DNA Glycosylase in Leishmania donovani Confers Tolerance against Antileishmanial Drugs.

Leishmania donovani is an intracellular protozoan parasite that causes endemic tropical disease visceral leishmaniasis (VL). Present drugs used against this fatal disease are facing resistance and toxicity issues. Survival of leishmania inside the host cells depends on the parasite's capacity to cope up with highly oxidative environment. Base excision repair (BER) pathway in L. donovani remains unexplored. We studied uracil DNA glycosylase (UNG), the key enzyme involved in BER pathway, and found that the glycosylase activity of recombinant LdUNG (Leishmania donovani UNG) expressed in E. coli is in sync with the activity of the parasite lysate under different reaction conditions. Overexpression of UNG in the parasite enhances its tolerance towards various agents which produce reactive oxygen species (ROS) and shows a higher infectivity in macrophages. Surprisingly, exposure of parasite to amphotericin B and sodium antimony gluconate upregulates the expression of UNG. Further, we found that the drug resistant parasites isolated from VL patients show higher expression of UNG. Mechanisms of action of some currently used drugs include accumulation of ROS. Our findings strongly suggest that targeting LdUNG would be an attractive therapeutic strategy as well as potential measure to tackle the problem of drug resistance in the treatment of leishmaniasis.

Vicinal abasic site impaired processing of a Tg:G mismatch and 8-oxoguanine lesions in three-component bistranded clustered DNA damage.

The occurrence of 7,8-dihydro-8-oxo-2'deoxyguanosine (8-oxodG), thymine glycol:guanine (Tg:G) mismatch and abasic site DNA damage lesions in close proximity induce repair refractive multicomponent clustered DNA damage. Herein, the influence of abasic sites in the processing of 8-oxodG lesion and Tg:G mismatch bistranded cluster is evaluated. Abasic sites are found to impart conformational destabilization that appreciably hinders the repair activity of the other lesions whenever present in a cluster combination. The repair process reduces the formation of double strand breaks (DSBs) and renders this three-lesion combination a non-DSB forming cluster. The stability of the DNA duplex harbouring these three lesions is highly compromised due to altered base helicity and base stacking phenomena leading to impaired repair.

Isolated coronary artery ectasia: Clinical, angiographic, and follow up characteristics.

BACKGROUND: Isolated Coronary artery ectasia (CAE) is considered an uncommon angiographic finding with varying patterns of presentation and carries significant morbidity burden to the patient. Our objective was to evaluate the prevalence of this condition, to analyse its clinical, angiographic, and follow up characteristics. PATIENTS AND METHODS: Coronary angiography was performed in 4950 patients from January 2009 to August 2014. The epidemiological, clinical, angiographic, and follow up characteristics of 52 patients with isolated CAE were examined. RESULTS: Of the 4950 angiograms analysed, isolated CAE was found in 52 patients, a prevalence of 1.05 %. The mean age of patients was 53.4 years. A predominance of the male sex was observed (71.1%). Angina on exertion was the most common presenting symptom (61.5%). Single vessel was involved in 61.5%. Left anterior descending artery was the most commonly involved vessel followed by right coronary artery, left circumflex and left main coronary artery. Type IV CAE as per Markis classification was the most common involvement. The median follow-up was 28±20 months, during which 10 patients (19.2%) had recurrent chest pain, and four patients were re-hospitalised, three for unstable angina, one for myocardial infarction. CONCLUSION: The prevalence of isolated coronary ectasia was 1.05%. The majority of patients had single vessel involvement, and left anterior descending branch was the most common involved vessel. This condition may not be considered completely benign, as it is associated with atherosclerotic risk factors and occurrence of coronary events including angina and myocardial infarction.

Microalbuminuria: Correlation With Prevalence and Severity of Coronary Artery Disease in Non-Diabetics.

BACKGROUND: Previous studies have shown that microalbuminuria (MAU) is an independent risk factor for cardiovascular diseases in diabetics, hypertensive patients and in the general population. However, the correlation of MAU with the severity of coronary artery disease (CAD) in non-diabetic patients has not been addressed in detail. This study aimed to investigate the relationship between MAU and severity of angiographically confirmed CAD in non-diabetic patients. METHODS: This was a cross-sectional study, which included 90 non-diabetic patients with documented CAD by coronary angiography. The ratio of urine albumin to creatinine was used to define MAU and severity of CAD was estimated using SYNTAX score. Patients were divided into two groups: group I that included patients without MAU and group II that included patients with MAU. RESULTS: Out of 90 non-diabetic CAD patients, 62 (68.9%) were in group I (MAU negative) and 28 (31.1%) were in group II (MAU positive). There was statistically significant difference in the median SYNTAX score between the groups (21 vs. 28, P < 0.001). The prevalences of double vessel CAD and triple vessel CAD were significantly higher in MAU positive group. There was a strong relationship between the presence of MAU and the extent and complexity of CAD (r = 0.094; P < 0.001). CONCLUSION: Thus, we conclude that patients with MAU have more severe angiographically detected CAD than those without MAU, and MAU exhibits a significant association with the presence and severity of CAD.

Effect of Ranolazine in Patients with Chest Pain and Normal Coronaries- A Hospital Based Study.

INTRODUCTION: There is an important role of coronary microcirculation in the clinical presentation and prognosis of patients who have typical chest pain despite normal epicardial coronary arteries (microvascular angina). Treatment of these patients is empirical because of the incomplete knowledge of its cause. Limited data has shown that ranolazine reduces angina and improves exercise performance in such patients with frequent angina. AIM: To evaluate the effect of ranolazine in patients with chest pain and normal epicardial coronaries (micro-vascular angina). MATERIALS AND METHODS: Sixty-five patients with anginal symptoms with abnormal exercise stress test and normal epicardial coronaries were enrolled for the study. All participants had baseline demographic and health history questionnaires, including Seattle Angina Questionnaire (SAQ) and Duke Activity Status Index (DASI). After enrolment, patients were randomly divided into two groups. One group (group 1) was assigned to ranolazine for six weeks along with other indicated anti-anginal drugs. The other group (group 2) was assigned to anti-anginal drugs other than ranolazine. Patients were reassessed for symptomatic and functional improvement (SAQ, DASI) at six weeks. RESULTS: Mean age of patients examined were 49.03 years in group 1 and 49.77 years in group 2. Approximately 42.9% of patients in group 1 and 40% in group 2 were male. Despite current anti-anginal therapy, patients in both the groups were symptomatic. At six weeks, 60% of patients in group 1 had angina as compared to 88.6% at baseline (p<0.05). Similarly, scores of domains of SAQ were higher at six weeks as compared to baseline (p<0.05) except for treatment satisfaction. No improvement of DASI score and functional capacity were seen in either group at six weeks as compared to baseline (p>0.05). At six weeks, angina was significantly lower in group 1 as compared to group 2 (60 % vs 86.7%; p<0.05). Four out of five SAQ subscale score were higher in ranolazine group as compared to the other group (p<0.05). Treatment satisfaction trended lower on ranolazine group (p<0.05). There was no significant differences in DASI in the two groups (DASI score 30.59 vs 29.85, p>0.05). CONCLUSION: Ranolazine is safe and improves symptoms significantly in patients with micro-vascular angina.

HAT2 mediates histone H4K4 acetylation and affects micrococcal nuclease sensitivity of chromatin in Leishmania donovani.

Histone post-translational modifications (PTMs) such as acetylation and methylation are known to affect chromatin higher order structures. Primary targets of these modifications include basic residues present at N-terminus tail region of core histones. Four histone acetyltransferase (HAT) genes have been identified in trypanosomatids. HAT1, HAT3 and HAT4 of Leishmania donovani have been partially characterized. However, there is no report about HAT2 of Leishmania donovani. Lysine residues present on the N-terminal tail of Leishmania donovani histone H4 are conserved in other trypanosomatids and humans. PTMs of lysines modulate various functions at chromatin level. The four histone acetyltransferases encoded in Leishmania genome were over-expressed to analyse their functional activity. All four HATs were found actively acetylating core histones H3/H4. Similar to L. donovani HAT3 and HAT4, HAT2 was found to be a member of MYST family protein and have SAS2 type domain. Over-expression of HAT2 significantly increases acetylation of H4K4. To analyse the effect of HAT2 over-expression on chromatin accessibility, micrococcal nuclease digestion assay was performed. MNase digestion resulted in a higher proportion of the mononucleosomes and dinucleosomes in HAT2 over-expressing cells as compared to WT L. donovani cells. Acetylation of lysine-4 neutralizes the amino terminal region of histone H4. This weakens its interaction with neighbouring nucleosomes and the linker DNA. HAT2 over-expression in L. donovani resulted in highly accessible chromatin suggesting chromatin decondensation. HAT2 may have an important role to play in global regulation of transcription in L. donovani. Better understanding of these epigenetic determinants of parasite would help in designing novel therapeutic strategies.

Idiopathic dilatation of pulmonary artery: A review.

The diagnosis of idiopathic dilatation of pulmonary artery is challenging because its clinical recognition is difficult and various other causes of dilated pulmonary artery need to be excluded. The clinical findings mimic various common cardiac disorders and both invasive and non-invasive investigations should be done to arrive at the diagnosis. It is a known clinical entity but etiology and pathophysiology are largely unknown. The current echocardiographic and catheterization based diagnostic criteria, may not be satisfied completely in a particular patient and need to be revisited in view of newer imaging modalities. There is paucity of information about the natural history of the disease with attendant lack of clarity in treatment guidelines. Certain cases may progress to huge dilatation and consequent serious implications. It is a rare disease and is the diagnosis of exclusion.

Plasma Gelsolin Level in HIV-1-Infected Patients: An Indicator of Disease Severity.

Plasma gelsolin (pGSN) is a multifunctional protein involved mainly in severing and clearing of actin filaments. Its level correlates with inflammation and several diseases making it a potential biomarker of diagnostic and prognostic values. The pGSN level in groups of treated and untreated HIV-1-infected Indian patients is investigated in this study. This study aims at investigating the levels of pGSN in HIV-1-infected patients across different age, sex, severity of disease, and treatment status. Blood samples of 213 patients were analyzed for CD4 counts by flow cytometry and pGSN was quantified by enzyme-linked immunosorbent assay (ELISA). The level of pGSN is significantly increased in HIV-1 infected patients (227.2 ± 54.3 µg/ml) compared to healthy volunteers (167.9 ± 61.8 µg/ml). The level correlates with CD4 cell counts as patients with lower CD4 counts showed higher pGSN levels and vice versa. Gender does not affect pGSN level; however, antiretroviral (ARV) treatment reduces pGSN toward normal. Within low CD4 cell count group, the untreated patients have 52% higher pGSN than healthy volunteers, whereas with treatment, the difference reduces to 24%. Similarly, high CD4 cell count (>350 cells/mm3) group of patients showed 44% increase in pGSN in untreated patients compared to 21% increase in treated patients. There is an upregulation of pGSN in HIV-1 infection and it is inversely correlated with CD4 cell counts. Treatment with ARV drugs decreases pGSN levels toward normal. The monitoring of pGSN level in HIV-1-infected patients could be an important indicator of severity of disease and recovery during treatment.

A quantum dot-MUC1 aptamer conjugate for targeted delivery of protoporphyrin IX and specific photokilling of cancer cells through ROS generation.

Non-targeted photosensitizers lack selectivity that undermines the potential use of photodynamic therapy (PDT). Herein, we report the DNA mediated assembly of a ZnSe/ZnS quantum dot (QD)-photosensitizer (PS)-Mucin 1(MUC1) aptamer conjugate for targeting the MUC1 cancer biomarker and simultaneous generation of reactive oxygen species (ROS). A photosensitizer, protoporphyrin IX (PpIX), was conjugated to a single stranded DNA and self-assembled to a complementary strand that was conjugated to a QD and harboring a MUC1 aptamer sequence. A multistep fluorescence resonance energy transfer (FRET) is shown that involves the QD, PpIX and covalently linked CF™ 633 amine dye (CF dye) to the MUC1 peptide that tracks the potency of the aptamer to attach itself with the MUC1 peptide. Since the absorption spectra of the CF dye overlap with the emission spectra of PpIX, the former acts as an acceptor to PpIX forming a second FRET pair when the dye labeled MUC1 binds to the aptamer. The binding of the QD-PpIX nanoassemblies with MUC1 through the aptamer was further confirmed by gel electrophoresis and circular dichroism studies. The selective photodamage of MUC1 expressing HeLa cervical cancer cells through ROS generation in the presence of the QD-PpIX FRET probe upon irradiation is successfully demonstrated.

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani.

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.

L-Asparaginase as a new molecular target against leishmaniasis: insights into the mechanism of action and structure-based inhibitor design.

l-Asparaginases belong to a family of amidohydrolases that catalyze the conversion of l-asparagine into l-aspartic acid and ammonia. Although bacterial l-asparaginases have been used extensively as anti-leukemic agents, their possible role as potential drug targets for pathogenic organisms has not been explored. The presence of genes coding for putative l-asparaginase enzymes in the Leishmania donovani genome hinted towards the specific role of these enzymes in extending survival benefit to the organism. To investigate whether this enzyme can serve as a potential drug target against the Leishmania pathogen, we obtained structural models of one of the putative Leishmania l-asparaginase I (LdAI). Using an integrated computational approach involving molecular modelling, docking and molecular dynamics simulations, we found crucial differences between catalytic residues of LdAI as compared to bacterial l-asparaginases. The deviation from the canonical acid-base pair at triad I, along with the structural reorganization of a ß-hairpin loop in the presence of a substrate, indicated an altogether new mechanism of action of the LdAI enzyme. Moreover, the finding of compositional and functional differences between LdAI and human asparaginase was used as a criterion to identify specific small molecule inhibitors. Through virtual screening of a library of 11 438 compounds, we report five compounds that showed favorable interactions with the active pocket of LdAI, without adversely affecting human asparaginase. One of these compounds when tested on cultured Leishmania promastigotes displayed a promising leishmanicidal effect. Overall, our work not only provides first hand mechanistic insights of LdAI but also proposes five strongly active compounds which may prove as effective anti-leishmaniasis molecules.

Squamous Cell Carcinoma of Lung Atypically Involving Heart: A Case Report With Literature Review.

Cardiac metastasis usually appears in patients with disseminated tumor disease. Involvement of heart in malignancy is generally underestimated and found to be in up to 25% of post mortem patients who had died of cancer. Cardiac involvement in metastases is usually uncommon; however, it may present with tachycardia, arrhythmia, cardiomegaly, heart failure, dyspnoea, hypotension, and pulsus paradoxus. Right side of heart is commonly known to be involved and the order of frequency of malignancies to metastasize to layers of the heart is pericardium, myocardium and endocardium.