Probing kinetic drug binding mechanism in voltage-gated sodium ion channel: open state versus inactive state blockers

The kinetics and nonequilibrium thermodynamics of open state and inactive state drug binding mechanisms have been studied here using different voltage protocols in sodium ion channel. We have found that for constant voltage protocol, open state block is more efficient in blocking ionic current than inactive state block. Kinetic effect comes through peak current for mexiletine as an open state blocker and in the tail part for lidocaine as an inactive state blocker. Although the inactivation of sodium channel is a free energy driven process, however, the two different kinds of drug affect the inactivation process in a different way as seen from thermodynamic analysis. In presence of open state drug block, the process initially for a long time remains entropy driven and then becomes free energy driven. However in presence of inactive state block, the process remains entirely entropy driven until the equilibrium is attained. For oscillating voltage protocol, the inactive state blocking is more efficient in damping the oscillation of ionic current. From the pulse train analysis it is found that inactive state blocking is less effective in restoring normal repolarisation and blocks peak ionic current. Pulse train protocol also shows that all the inactive states behave differently as one inactive state responds instantly to the test pulse in an opposite manner from the other two states.     

Enzymatic attributes of an l-isoaspartyl methyltransferase from Candida utilis and its role in cell survival

BackgroundsSpontaneous deamidation and isoaspartate (IsoAsp) formation contributes to aging and reduced longevity in cells. A protein-l-isoaspartate (d-aspartate) O-methyltransferase (PCMT) is responsible for minimizing IsoAsp moieties in most organisms.MethodsPCMT was purified in its native form from yeast Candida utilis. The role of the native PCMT in cell survival and protein repair was investigated by manipulating intracellular PCMT levels with Oxidized Adenosine (AdOx) and Lithium Chloride (LiCl). Proteomic Identification of possible cellular targets was carried out using 2-dimensional gel electrophoresis, followed by on-Blot methylation and mass spectrometric analysis.ResultsThe 25.4 kDa native PCMT from C. utilis was found to have a K_m of 3.5 µM for AdoMet and 33.36 µM for IsoAsp containing Delta Sleep Inducing Peptide (DSIP) at pH 7.0. Native PCMT comprises of 232 amino acids which is coded by a 698 bp long nucleotide sequence. Phylogenetic comparison revealed the PCMT to be related more closely with the prokaryotic homologs. Increase in PCMT levels in vivo correlated with increased cell survival under physiological stresses. PCMT expression was seen to be linked with increased intracellular reactive oxygen species (ROS) concentration. Proteomic identification of possible cellular substrates revealed that PCMT interacts with proteins mainly involved with cellular housekeeping. PCMT effected both functional and structural repair in aged proteins in vitro.General significanceIdentification of PCMT in unicellular eukaryotes like C. utilis promises to make investigations into its control machinery easier owing to the familiarity and flexibility of the system.     

Vaccination with Human Papillomavirus Pseudovirus-Encapsidated Plasmids Targeted to Skin Using Microneedles

Human papilloma virus-like particles (HPV VLP) serve as the basis of the current licensed vaccines for HPV. We have previously shown that encapsidation of DNA expressing the model antigen M/M2 from respiratory syncytial virus (RSV) in HPV pseudovirions (PsV) is immunogenic when delivered intravaginally. Because the HPV capsids confer tropism for basal epithelium, they represent attractive carriers for vaccination targeted to the skin using microneedles. In this study we asked: 1) whether HPV16 VLP administered by microneedles could induce protective immune responses to HPV16 and 2) whether HPV16 PsV-encapsidated plasmids delivered by microneedles could elicit immune responses to both HPV and the antigen delivered by the transgene. Mice immunized with HPV16 VLP coated microneedles generated robust neutralizing antibody responses and were protected from HPV16 challenge. Microneedle arrays coated with HPV16-M/M2 or HPV16-F protein (genes of RSV) were then tested and dose-dependent HPV and F-specific antibody responses were detected post-immunization, and M/M2-specific T-cell responses were detected post RSV challenge, respectively. HPV16 PsV-F immunized mice were fully protected from challenge with HPV16 PsV and had reduced RSV viral load in lung and nose upon intranasal RSV challenge. In summary, HPV16 PsV-encapsidated DNA delivered by microneedles induced neutralizing antibody responses against HPV and primed for antibody and T-cell responses to RSV antigens encoded by the encapsidated plasmids. Although the immunogenicity of the DNA component was just above the dose response threshold, the HPV-specific immunity was robust. Taken together, these data suggest microneedle delivery of lyophilized HPV PsV could provide a practical, thermostable combined vaccine approach that could be developed for clinical evaluation.     

Molecular Pathogenesis of Wilson Disease Among Indians: A Perspective on Mutation Spectrum in ATP7B gene, Prevalent Defects, Clinical Heterogeneity and Implication Towards Diagnosis

Aims We aim to identify the molecular defects in the ATP7B, the causal gene for Wilson disease (WD), in eastern Indian patients and attempt to assess the overall mutation spectrum in India for detection of mutant allele for diagnostic purposes. Methods Patients from 109 unrelated families and their first-degree relatives comprising 400 individuals were enrolled in this study as part of an ongoing project. Genomic DNA was prepared from the peripheral blood of Indian WD patients. PCR was done to amplify the exons and flanking regions of the WD gene followed by sequencing, to identify the nucleotide variants. Results In addition to previous reports, we recently identified eight mutations including three novel (c.3412 + 1G > A, c.1771 G > A, c.3091 A > G) variants, and identified patients with variable phenotype despite similar mutation background suggesting potential role of modifier locus. Conclusions So far we have identified 17 mutations in eastern India including five common mutations that account for 44% of patients. Comparative study on WD mutations between different regions of India suggests high genetic heterogeneity and the absence of a single or a limited number of common founder mutations. Genotype–phenotype correlation revealed that no particular phenotype could be assigned to a particular mutation and even same set of mutations in different patients showed different phenotypes.     

Effects of salt and osmotic shocks on unadapted and adapted callus lines of tobacco

Growth, viability and proline content of adapted and unadapted calluses of Nicotiana tabacum L. var. Jayasri, affected due to osmotic stresses and particularly to stress-shocks treated with different osmotica like NaCl (ionic-penetrating), mannitol (non-ionic-penetrating) and polyethylene glycol, (PEG) (non-ionic-non penetrating) were studied to evaluate the physiological differences of stress effects. The tissues adapted to a low concentration of NaCl (85 mM) showed low growth with high proline content compared to the tissues adapted to a low concentration of mannitol (165 mM). Proline content was similar in tissues adapted to high concentrations of NaCl (171 mM) and mannitol (329 mM) but growth in the latter case was relatively low. Growth and viability were subsequently correlated with the pattern of retention in or diffusion of proline out of the tissues after shock-treatments. The loss of tissue viability of the adapted calluses was comparatively less than the unadapted callus even after shock-treatments with 1282 mM NaCl and 823 mM mannitol. The former calluses retained the capability of regrowth though at a slow rate. Such adapted tissues also retained more proline. The mannitol-adapted tissues, when shocked with PEG (200 g l-1), showed low viability with more diffusion and a very little retention of proline while, in the unadapted tissue, all the proline was leached out. The results indicated that the effects of different osmotica on plant tissue varied depending upon the physico-chemical nature of the compounds used as stress-inducing-agents, and retention and diffusion of proline was altered when the tissues were shocked with high concentrations of all these compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of HCV genotypes and HCV RNA viral load in hepatitis infected patients of Kolar region,Karnataka, India

Decisions and disease progression prediction, understanding the distribution of the hepatitis C virus (HCV) genotype and its association with viral load is significant for treatment. Therefore, it is of interest to document the distribution pattern of HCV genotypes and their association with viral load among HCV infected patients in Kolar, Karnataka. Seventy-four HCV-positive patients and not on antiviral therapy were enrolled from R.L. Jalappa hospital in Kolar, Karnataka. Blood samples were taken and demographics were recorded. HCV RNA was isolated after plasma was separated. qPCR was performed to measure the viral load, and RT-PCR was performed to determine the genotype. Genotype 3 was the prevalent (n=11, 40.7%) followed by genotype 4 (n=8, 29.6%), 2 (n=6, 22.2%), 1 (n= 13.7%), and mix (n=1, 13.7%). The median viral load of genotype 3 was a 2,87,835 IU/mL (IQR 10, 780-3, 71, 66) , genotype 2 was 81,030 IU/mL (IQR 66,495-95,565), genotype 4 was 43, 410 IU/mL (IQR 38, 355-48, 465) belongs to viral load less than 8,00,000 IU/mL. The median viral load genotype 3 was a 1, 05, 19, 500 IU/mL (IQR 49, 37, 250-2, 36, 71, 500), genotype 2 was 2,55,99,000 IU/mL (IQR 2,00,10,000-32,725,500), genotype 4 was 1,67,40,000 IU/mL (IQR 1,45,50,000-17,493,000) belonging to viral load more than 8,00,000 IU/mL category. A correlation between genotype and viral load was observed (p =1.5x10-12), of which genotype 3 showed a high viral load. Thus, HCV genotypes 1 2, 3, 4, and mixed genotype was observed in the patients studied. HCV genotype was associated with viral load in patient plasma. This data finds use in the treatment and prevention of hepatitis C in Kolar, Karnataka.     

Height, weight and earnings among coalminers in India

This paper analyses earning/wage differentials by height among coalmine workers in India. Our findings suggest that workers of above average height earn 9-17% more than their shorter counterparts and 6-13% more than average reference height. The results suggest that long-term investments in health human capital might ensure increase of labour productivity and thereby earnings, particularly in underdeveloped economies.     

Improved patient-reported outcomes in patients with psoriatic arthritis treated with abatacept: results from a phase 3 trial

Background

To explore the effect of abatacept treatment on patient-reported outcomes (PROs) in psoriatic arthritis (PsA).

Methods

Patients with PsA were randomised (1:1) to subcutaneous abatacept 125?mg weekly/placebo for 24?weeks with early escape (EE) to open-label abatacept (week 16). Adjusted mean changes from baseline to weeks 16 (all patients) and 24 (non-EE responders) in Health Assessment Questionnaire-Disability Index (HAQ-DI), Short Form-36 (SF-36; physical and mental component summary and domains), Dermatology Life Quality Index and Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) were evaluated. Subpopulations were analysed by baseline C-reactive protein (CRP) level (> vs?≤?upper limit of normal [ULN]) and prior tumour necrosis factor inhibitor (TNFi) exposure. Proportions of patients reporting improvements ≥ minimal clinically important differences (MCIDs) and?≥?normative values (NVs) in HAQ-DI, SF-36 and FACIT-F (week 16 before EE) were analysed.

Results

In total population, numerically higher improvements in most PROs were reported with abatacept (n?=?213) versus placebo (n?=?211) at both time points (P?>?0.05). Higher proportions of abatacept versus placebo patients reported PRO improvements ≥ MCID and?≥?NV at week 16. At week 16, all PRO improvements were numerically greater (P?>?0.05) in patients with baseline CRP?>?ULN versus CRP?≤?ULN (all significant [95% confidence interval] for abatacept vs placebo); improvements in SF-36 component summaries and FACIT-F were greater in TNFi-naïve versus TNFi-exposed patients (abatacept > placebo). Week 24 subgroup data were difficult to interpret due to low patient numbers.

Conclusions

Abatacept treatment improved PROs in patients with PsA versus placebo, with better results in elevated baseline CRP and TNFi-naïve subpopulations.

Trial registration

ClinicalTrials.gov number, NCT01860976 (funded by Bristol-Myers Squibb); date of registration: 23 May 2013.