Netarsudil monotherapy as the initial treatment for open-angle glaucoma and ocular hypertension in Indian patients: A real-world evaluation of efficacy and safety.

Purpose: Glaucoma is the second leading cause of blindness worldwide, affecting more than 64 million people aged 40-80. The best way to manage primary open-angle glaucoma (POAG) is by lowering the intraocular pressure (IOP). Netarsudil is a Rho kinase inhibitor, the only class of antiglaucoma medications that reorganizes the extracellular matrix to improve the aqueous outflow through the trabecular pathway. Methods: An open-label, real-world, multicentric, observation-based 3-month study was performed for assessing the safety and ocular hypotensive efficacy of netarsudil ophthalmic solution (0.02% w/v) in patients with elevated IOP. Patients were given netarsudil ophthalmic solution (0.02% w/v) as a first-line therapy. Diurnal IOP measurements, best-corrected visual acuity, and adverse event assessments were recorded at each of the five visits (Day-1: screening day and first dosing day; subsequent observations were taken at 2 weeks, 4 weeks, 6 weeks, and 3 months). Results: Four hundred and sixty-nine patients from 39 centers throughout India completed the study. The mean IOP at baseline of the affected eyes was 24.84 ± 6.39 mmHg (mean ± standard deviation). After the first dose, the IOP was measured after 2, 4, and 6 weeks, with the final measurement taken at 3 months. The percentage reduction in IOP in glaucoma patients after 3 months of once-daily netarsudil 0.02% w/v solution use was 33.34%. The adverse effects experienced by patients were not severe in the majority of cases. Some adverse effects observed were redness, irritation, itching, and others, but only a small number of patients experienced severe reactions, as reported in a decreasing order: redness > irritation > watering > itching > stinging > blurring. Conclusion: We found that netarsudil 0.02% w/v solution monotherapy when used as the first-line treatment in primary open-angle glaucoma and ocular hypertension was both safe and effective.

A novel nucleic acid extraction method from aromatic herbs and dried herbal powders using cow skim milk.

Authenticity of dried aromatic herbs and herbal powders for the ASU (ayurvedic, siddha, unani) drug formulations is a key of their clinical success. The DNA based authentication is an answer; however, extraction of PCR quality DNA from such material is often problematic due to the presence of various co-extracted PCR inhibitors. Here, we report a novel DNA isolation and purification method utilizing cow skim milk that successfully yields PCR quality DNA from the aromatic herbs and dried herbal powders. The improved method presented in this study could be used as an alternative to successfully extract PCR quality DNA from such plant materials. Further, we present a set of robust matK primers which could be used as plant barcoding resource in future studies.

Ancient mtDNA from the extinct Indian cheetah supports unexpectedly deep divergence from African cheetahs.

The Indian cheetah was hunted to extinction by the mid-20th century. While analysis of 139 bp of mitochondrial DNA (mtDNA) has confirmed that the Indian cheetah was part of the Asiatic subspecies (Acinonyx jubatus venaticus), the detailed relationships between cheetah populations remains unclear due to limited genetic data. We clarify these relationships by studying larger fragments of cheetah mtDNA, both from an Indian cheetah museum specimen and two African cheetah, one modern and one historic, imported into India at different times. Our results suggest that the most recent common ancestor of cheetah mtDNA is approximately twice as ancient as currently recognised. The Indian and Southeast African (Acinonyx jubatus jubatus) cheetah mtDNA diverged approximately 72 kya, while the Southeast and Northeast African (Acinonyx jubatus soemmeringii) cheetah mtDNA diverged around 139 kya. Additionally, the historic African cheetah sampled from India proved to have an A. j. jubatus haplotype, suggesting a hitherto unrecognised South African route of cheetah importation into India in the 19th century. Together, our results provide a deeper understanding of the relationships between cheetah subspecies, and have important implications for the conservation of A. j. venaticus and potential reintroduction of cheetahs into India.

Incidence and predictors of pacemaker-induced cardiomyopathy with comparison between apical and non-apical right ventricular pacing sites.

BACKGROUND: Asynchronous activation of left ventricle (LV) due to chronic right ventricular (RV) pacing has been known to predispose to LV dysfunction. The predictors of LV dysfunction remain to be prospectively studied. This study was designed to follow up patients with RV pacing to look for development of pacing-induced cardiomyopathy (PiCMP), identify its predictors and draw comparison between apical vs non-apical RV pacing sites. METHODS: Three hundred sixty-three patients undergoing dual-chamber and single-chamber ventricular implants were enrolled and followed up. Baseline clinical parameters; paced QRS duration and axis; RV lead position by fluoroscopy; LV ejection fraction (LVEF) by Simpson's method on transthoracic echocardiography (TTE); intraventricular dyssynchrony (septal-posterior wall contraction delay) and interventricular dyssynchrony (aortopulmonary ejection delay) on TTE were recorded. The patients were followed up at 6-12 monthly interval with estimation of LVEF and pacemaker interrogation at each visit. Pacemaker-induced cardiomyopathy (PiCMP) was defined as a fall in ejection fraction of 10% as compared to the baseline LVEF. Patients developing PiCMP were compared to other patients to identify predictors. RESULTS: The mean age of study population was 59.8 years, 68.3% being males. Fifty-one percent and 49% patients underwent VVIR and DDDR pacemaker implantation, respectively. After attrition, 254 patients were analysed. PiCMP developed in 35 patients (13.8%) over a mean follow-up of 14.5 months. After multivariate analysis, burden of ventricular pacing > 60% [HR 4.26, p = 0.004] and interventricular dyssynchrony (aortopulmonary ejection delay > 40 msec) [HR 3.15, p = 0.002] were identified as predictors for PiCMP in patients undergoing chronic RV pacing. There was no effect of RV pacing site (apical vs non-apical) on incidence of PiCMP [HR 1.44, p = 0.353). CONCLUSIONS: Incidence of PiCMP with RV pacing was found to be 13.8% over a mean follow-up of 14.5 months. Burden of right ventricular pacing and interventricular dyssynchrony were identified as the most important predictors for the development of PiCMP. Non-apical RV pacing site did not offer any benefit in terms of incidence of PiCMP over apical lead position.

Pulmonary artery from the left main coronary artery.

The usual sources of pulmonary blood flow in pulmonary atresia (PA) with(VSD) are patent ductus arteriosus and aortopulmonary collaterals. However, rarely fistulous collaterals may also arise from the coronary arteries which usually open into the main pulmonary trunk or branch pulmonary arteries. In such cases, selective coronary angiogram may be required for the demonstration of pulmonary arterial anatomy. A case of PA with VSD with failure to demonstrate pulmonary arteries on routine catheterization study (ventricular, aortic root, and descending aortic angiograms) is being presented here. A coronary artery-to-pulmonary artery fistula was suspected in view of dilated left main coronary artery, and pulmonary arteries were well demonstrated with selective coronary angiogram.

Mechanism of Mg2+-Accompanied Product Release in Sugar Nucleotidyltransferases.

The nucleotidyl transfer reaction, catalyzed by sugar nucleotidyltransferases (SNTs), is assisted by two active site Mg2+ ions. While studying this reaction using X-ray crystallography, we captured snapshots of the pyrophosphate (product) as it exits along a pocket. Surprisingly, one of the active site Mg2+ ions remains coordinated to the exiting pyrophosphate. This hints at the participation of Mg2+ in the process of product release, besides its role in catalyzing nucleotidyl transfer. These observations are further supported by enhanced sampling molecular dynamics simulations. Free energy computations suggest that the product release is likely to be rate limiting in SNTs, and the origin of the high free energy barrier for product release could be traced back to the "slow" conformational change of an Arg residue at the exit end of the pocket. These results establish a dual role for Mg2+, and propose a general mechanism of product release during the nucleotidyl transfer by SNTs.

Primary percutaneous coronary intervention for acute ST elevation myocardial infarction: Outcomes and determinants of outcomes: A tertiary care center study from North India.

BACKGROUND: Primary percutaneous coronary intervention (PCI) is the current standard of care for acute ST elevation myocardial infarction (STEMI). Most of the data on primary PCI in acute STEMI is from western countries. We studied the outcomes of primary PCI for acute STEMI at a tertiary care center in North India. METHODS: Consecutive patients undergoing primary PCI for STEMI were prospectively studied during the period from February 2103 to May 2015. The outcomes assessed were all cause in hospital mortality, factors associated with mortality, major adverse cardiac and cerebrovascular event rate (composite of all cause in hospital mortality, non-fatal re infarction and stroke) and procedural complications. RESULTS: 371 patients underwent primary PCI during the study period. The mean age was 54 years and 82.7% were males. The mean total ischemia time and door to balloon times were 6.8h and 51min respectively. 96.4% patients underwent successful primary PCI. The total in hospital mortality was 12.9%. Mortality with cardiogenic shock at presentation was 66.7% while non-shock mortality was 2.6%. In hospital MACCE rate was 13.5%. Factors significantly associated with mortality were KILLIP class (OR: 8.4), door to balloon time (OR 1.02), final TIMI flow (OR 0.44) and severe LV dysfunction (OR 22.0). Procedure related adverse events were rare and there was no non-CABG associated major TIMI bleeding. CONCLUSION: Primary PCI for acute STEMI is feasible in our setup and associated with high success rate, low mortality in non-shock patients and low complication rates.

The evaluation of serum amylase in the patients of type 2 diabetes mellitus, with a possible correlation with the pancreatic functions.

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder which is associated with hyperglycaemia. It is caused by a derangement in the secretion or function of the endocrinal portion of the pancreas. There is a close anatomical and functional relationship between its exocrine and endocrine portions. To address this issue, the current study was designed to evaluate the blood glucose and the amylase levels of diabetic patients as representatives of the two portions of the pancreas respectively. AIMS AND OBJECTIVES: The aim of the present study was to determine the blood glucose, serum amylase and the serum lipid profile in known cases of type 2 Diabetes Mellitus and to compare and correlate these parameters with those of age and sex matched healthy controls. MATERIAL AND METHODS: One hundred ten patients of type 2 Diabetes mellitus, who were already diagnosed and were taking treatment, were included in this study. 30 age and sex matched healthy individuals were recruited as the control group in our study. Fasting venous blood samples were collected from the patients as well as the controls and they were analysed by using an automated analyser for blood glucose, serum amylase and the lipid profile (serum triglycerides, total cholesterol, high density lipoproteins and low density lipoproteins). The results were analyzed statistically by using the Student's "t" test and correlation coefficients. RESULTS: Significantly low serum amylase levels were found in the diabetic patients as compared to those in the healthy controls (p value <0.001). Also, the levels of fasting serum total cholesterol, triglycerides and the low density lipoproteins were significantly higher in the patients as compared to those in the controls, with p values of <0.05, <0.001 and <0.001 respectively. The HDL (high density lipoprotein) level was found to be lower in the diabetic patients (p value <0.001). CONCLUSIONS: From our study, it was concluded that in type 2 Diabetes mellitus, wherever the blood glucose level was higher, the serum amylase activity was found to be significantly lower. This reflected the derangement in the endocrine-exocrine axis of the pancreas, as a disease which affected any portion of an organ would affect the adjoining area of that organ functionally. This fact must be kept in mind while the patients are treated.

Crystal structures identify an atypical two-metal-ion mechanism for uridyltransfer in GlmU: its significance to sugar nucleotidyl transferases.

N-Acetylglucosamine-1-phosphate uridyltransferase (GlmU), exclusive to prokaryotes, is a bifunctional enzyme that synthesizes UDP-GlcNAc-an important component of the cell wall of many microorganisms. Uridyltransfer, one of the reactions it catalyzes, involves binding GlcNAc-1-P, UTP and Mg(2+) ions; however, whether one or two ions catalyze this reaction remains ambiguous. Here, we resolve this using biochemical and crystallographic studies on GlmU from Mycobacterium tuberculosis (GlmU(Mtb)) and identify a two-metal-ion mechanism (mechanism-B). In contrast to well-established two-metal mechanism (mechanism-A) for enzymes acting on nucleic acids, mechanism-B is distinct in the way the two Mg(2+) ions (Mg(2+)A and Mg(2+)B) are positioned and stabilized. Further, attempts to delineate the roles of the metal ions in substrate stabilization, nucleophile activation and transition-state stabilization are presented. Interestingly, a detailed analysis of the available structures of sugar nucleotidyl transferases (SNTs) suggests that they too would utilize mechanism-B rather than mechanism-A. Based on this, SNTs could be classified into Group-I, which employs the two-metal mechanism-B as in GlmU, and Group-II that employs a variant one-metal mechanism-B, wherein the role of Mg(2+)A is substituted by a conserved lysine. Strikingly, eukaryotic SNTs appear confined to Group-II. Recognizing these differences may be important in the design of selective inhibitors against microbial nucleotidyl transferases.

Viral protein complexed liposomes for intranasal delivery of hepatitis B surface antigen.

The present work investigates prospective of recombinantly expressed influenza surface protein haemagglutinin (HA) complexed liposomes for intranasal delivery of HBsAg. Liposomes encapsulating HBsAg were prepared and complexed with HA. The prepared formulations were extensively characterized for vesicle size, polydispersity index, entrapment efficiency, HA complexation efficiency, in vitro release, etc. Stability of protein molecules was accessed by SDS-PAGE. The antigenicity of protein HBsAg was determined by EIA and the functional stability of HA was evaluated by haemagglutination assay. Subsequently, in vivo study was carried out to study their feasibility as nasal vaccine carriers. A significant and perdurable immune response was obtained following in vivo administration of the developed formulations that was comparable with alum adsorbed HBsAg administered intramuscularly. The HA complexed liposomal formulations elicited sIgA in mucosal secretions and also demonstrated cellular immune response both of which are not induced in the case of alum adsorbed HBsAg vaccine. Further, the HA complexed liposomes produced higher immune response as compared to plain liposomes that might be due to higher uptake of former as evidenced in microscopy study of nasal tissues. The higher cellular response generated by HA complexed liposomes may be possibly due to characteristic pH dependent fusion property of HA protein.

Pre- and post- donation haematological values in healthy donors undergoing plateletpheresis with five different systems.

BACKGROUND: Although automated cell separators have undergone a lot of technical refinements, attention has been focused more on the quality of platelet concentrates than on donor safety. We planned this prospective study to observe the effects of automated plateletpheresis on normal haematological values of healthy donors and to determine whether the haematological alterations had any clinical consequences. STUDY DESIGN AND METHODS: The study was conducted on 457 healthy, first-time plateletpheresis donors over a period of 26 months. The plateletpheresis procedures were performed using five different cell separators and various pre- and post-donation haematological values such as haemoglobin concentration (Hb), haematocrit (Hct), platelet and white blood cell (WBC) counts, mean platelet volume and platelet distribution width were measured in all donors. RESULTS: We observed that the Hb, Hct, platelet and WBC counts decreased significantly in the donors (p<0.01) after each procedure, without there being significant changes in mean platelet volume or platelet distribution width. The decreases in Hb and Hct were significantly greater with the CS 3000 and Amicus machines, while the decreases in platelet and WBC counts were significantly greater with the CS 3000 and Fresenius separators. CONCLUSION: Although a significant drop in complete blood count was observed in all donors, none manifested features of thrombocytopenia or anaemia. Nevertheless, more prospective studies on this aspect are required in order to establish guidelines for donor safety in apheresis and also to help in assessing donor suitability, especially given the present trend of double product apheresis collections.

Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.

Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues.

Structure of N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) from Mycobacterium tuberculosis in a cubic space group.

GlmU is a bifunctional enzyme that catalyzes the final two steps in the biosynthesis of UDP-GlcNAc. Crystals of GlmU from Mycobacterium tuberculosis obtained using ammonium sulfate as a precipitant diffracted poorly (to 3.4 A resolution) and displayed an unusually high solvent content (>80%) with sparse crystal packing that resulted in large solvent channels. With one molecule per asymmetric unit, the monomers from three neighbouring asymmetric units related by the crystal threefold formed a biological trimer. Although this is the first report of the structure of GlmU determined in a cubic crystal form, the trimeric arrangement here is similar to that observed for other GlmU structures determined in hexagonal (H3, H32, P6(3)22) space groups.

The significance of EXDD and RXKD motif conservation in Rel proteins.

Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3'-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg(2+) due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5'P(alpha)-O-P(beta) bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.

PknB-mediated phosphorylation of a novel substrate, N-acetylglucosamine-1-phosphate uridyltransferase, modulates its acetyltransferase activity.

Identifying direct targets of kinases and determining how their activities are regulated are central to understanding how they generate biological responses. Genetic and biochemical studies have shown that Mycobacterium tuberculosis serine/threonine protein kinases PknA and PknB play a role in modulating cell shape and possibly cell division. In this report, we show that the enzyme N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) of M. tuberculosis is a novel substrate of PknB and is phosphorylated on threonine residues. GlmU carries out two important biochemical activities: a C-terminal domain catalyzes the transfer of acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce N-acetylglucosamine-1-phosphate, which is converted into UDP-N-acetylglucosamine by the transfer of uridine 5'-monophosphate (from uridine 5'-triphosphate), a reaction catalyzed by the N-terminal domain. We determined the crystal structures of GlmU in apo form and UDP-N-acetylglucosamine-bound form, and analyzed them to identify threonine residues that may be accessible to PknB. The structure shows a two-domain architecture, with an N-terminal domain having an alpha/beta-like fold and with a C-terminal domain that forms a left-handed parallel beta-helix structure. Kinase assays with PknB using the N- and C-terminal domains of GlmU as substrates illustrated that PknB phosphorylates GlmU in the C-terminal domain. Furthermore, mutational studies reveal one of the five threonines present in region 414-439 to be phosphorylated by PknB. Structural and biochemical analyses have shown the significance of a variable C-terminal tail in regulating acetyltransferase activity. Notably, we demonstrate that although PknB-mediated phosphorylation of GlmU does not affect its uridyltransferase activity, it significantly modulates the acetyltransferase activity. These findings imply a role for PknB in regulating peptidoglycan synthesis by modulating the acetyltransferase activity of GlmU.

The pygmy hog is a unique genus: 19th century taxonomists got it right first time round.

The pygmy hog, Sus salvanius, the smallest and rarest extant suid was first described as the only member of the genus Porcula. It is currently regarded as member of the genus Sus and a sister taxon of the domestic pig/Eurasian wild boar (Sus scrofa). Phylogenetic analyses of 2316 bp from three mtDNA loci (control-region, cytochrome b, 16S) by Bayesian inference and statistical testing of alternative phylogenetic hypotheses all support the original classification of the pygmy hog as a unique genus. Thus, we propose that the species name Porcula salvania should be resurrected. The reclassification will heighten awareness of the need for the future protection and survival of this unique species.

Structural stabilization of GTP-binding domains in circularly permuted GTPases: implications for RNA binding.

GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1-G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4-G5-G1-G2-G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an 'anchoring' C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding.