Pillar cell and erythrocyte localization of fugu ET(A) receptor and its implication

Endothelin, a vasoconstrictor peptide, plays important roles not only in the mammalian circulatory system but also in non-mammalian systems, such as the gill lamellar vascular network with complex structural characteristics. Here, we show that (i) the contraction of pillar cells that delimit the lamellar vasculature is controlled by endothelin through the type A endothelin receptor (ET(A)) linked to the intracellular calcium signaling system and (ii) ET(A) receptor is also highly expressed on fugu erythrocytes, a hitherto unexpected finding. Database mining revealed the presence of five endothelin receptor (ETR) sequences in the fugu genome. By Northern blotting, cDNA cloning, and fura-2 monitoring, the branchial ETR subtype was shown to be ET(A) able to induce a Ca(2+) transit. Immunohistochemistry revealed its pillar cell and erythrocyte localization. These results suggest an endothelin/ET(A)-mediated coordinated regulation of the pillar cell shape and erythrocyte membrane flexibility.     

Regional Expression of Key Cell Cycle Proteins in Brain from Subjects with Amnestic Mild Cognitive Impairment

Mild cognitive impairment (MCI) is regarded as a transition stage between the cognitive changes of normal aging and the more serious problems caused by Alzheimer’s disease (AD). Previous studies had demonstrated increased expression of cell cycle proteins in AD brain. In the present study, we have analyzed the expression of the cell cycle proteins, CDK2, CDK5 and cyclin G1 in hippocampus and inferior parietal lobule (IPL) in subjects with amnestic mild cognitive impairment and control using Western blot analysis. The expression of CDK2, CDK5 and cyclin G1 were found to be significantly increased in MCI hippocampus as well as in IPL compared to control brain. These results suggest that some cells may have re-entered the cell cycle. However, the expression of CDK2 and CDK5 is greater in MCI hippocampus compared to those of MCI IPL, and hippocampus is a region that is severely affected by AD pathology. Since these proteins are involved directly or indirectly in microtubule destabilization and hyperphosphorylation of tau, and also in APP processing we hypothesize that cell cycle disturbance may be important contributor in the pathogenesis of AD. Special issue dedicated to Dr. John P. Blass.     

A novel multilocus sequence typing scheme identifying genetic diversity amongst Leishmania donovani isolates from a genetically homogeneous population in the Indian subcontinent

In the Indian subcontinent, infection with Leishmania donovani can cause fatal visceral leishmaniasis. Genetic variation in L. donovani is believed to occur rapidly from environmental changes and through selective drug pressures, thereby allowing continued disease occurrence in this region. All previous molecular markers that are commonly in use multilocus microsatellite typing and multilocus sequence typing, were monomorphic in L. donovani originating from the Indian subcontinent (with only a few exceptions) and hence are not suitable for this region. An multilocus sequence typing scheme consisting of a new set of seven housekeeping genes was developed in this study, based on recent findings from whole genome sequencing data. This new scheme was used to assess the genetic diversity amongst 22 autochthonous L. donovani isolates from Bangladesh. Nineteen additional isolates of the L. donovani complex (including sequences of L. donovani reference strain BPK282A1) from other countries were included for comparison. By using restriction fragment length polymorphism of the internal transcribed spacer 1 region (ITS1-RFLP) and ITS1 sequencing, all Bangladeshi isolates were confirmed to be L. donovani. Population genetic analyses of 41 isolates using the seven new MLST loci clearly separated L. donovani from Leishmania infantum. With this multilocus sequence typing scheme, seven genotypes were identified amongst Bangladeshi L. donovani isolates, and these isolates were found to be phylogenetically different compared with those from India, Nepal, Iraq and Africa. This novel multilocus sequence typing approach can detect intra- and inter-species variations within the L. donovani complex, but most importantly these molecular markers can be applied to resolve the phylogenetically very homogeneous L. donovani strains from the Indian subcontinent. Four of these markers were found suitable to differentiate strains originating from Bangladesh, with marker A2P being the most discriminative one.     

Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease

Parkinson's disease (PD) is an age‐related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α‐synuclein‐containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN‐induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early‐onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy‐lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2‐D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6‐month‐old PINK1‐deficient mice and wild‐type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD.

     

Genital Human Papillomavirus Infection among Women in Bangladesh: Findings from a Population-Based Survey

Background

There has been no population-based study on human papillomavirus (HPV) prevalence or its genotypes in Bangladesh; a country eligible for GAVI funding for HPV vaccine.

Methods

We used baseline survey data of a prospective cohort study that was conducted in one urban and one rural area of Bangladesh. A total of 997 urban and 905 rural married women, aged 13 to 64 years, were enrolled in the baseline during July-December, 2011. Information was collected on socio-demographic characteristics and potential risk factors for HPV infection followed by gynecological examination and collection of endocervical samples using the cervical cytobrush (Digene cervical sampler). HPV DNA testing was done by Polymerase Chain Reaction (PCR) using a consensus primer set.

Results

Prevalence of any HPV infection was 7.7% with no significant difference between urban and rural women. Most common high-risk genotypes were HPV16, HPV66, HPV18, HPV45, HPV31 and HPV53. Urban women working as housemaids or garment workers were at higher risk of any HPV infection (OR = 2.15, 95% CI: 1.13–4.11) compared to housewives. Rural women whose husband lived overseas were almost two times more likely to have any HPV infection (OR = 1.93; 95% CI 1.05–3.55) compared to women whose husbands lived with them.

Conclusion

The prevalence of HPV infection among Bangladeshi women is similar to other regions of Asia. However, type-specific patterns are different. The study findings will inform the formulation of HPV vaccination policies in Bangladesh, monitoring the impact of vaccination programmes, and the identification of target populations for screening.     

The complete mitochondrial genome sequence of Oncicola luehei (Acanthocephala: Archiacanthocephala) and its phylogenetic position within Syndermata

In the present study, we determined the complete mitochondrial genome sequence of Oncicola luehei (14,281bp), the first archiacanthocephalan representative and the second complete sequence from the phylum Acanthocephala. The complete genome contains 36 genes including 12 protein coding genes, 22 transfer RNA (tRNA) genes and 2 ribosomal RNA genes (rrnL and rrnS) as reported for other syndermatan species. All genes are encoded on the same strand. The overall nucleotide composition of O. luehei mtDNA is 37.7% T, 29.6% G, 22.5% A, and 10.2% C. The overall A+T content (60.2%) is much lower, compared to other syndermatan species reported so far, due to the high frequency (18.3%) of valine encoded by GTN in its protein-coding genes. Results from phylogenetic analyses of amino acid sequences for 10 protein-coding genes from 41 representatives of major metazoan groups including O. luehei supported monophyly of the phylum Acanthocephala and of the clade Syndermata (Acanthocephala+Rotifera), and the paraphyly of the clade Eurotatoria (classes Bdelloidea+Monogononta from phylum Rotifera). Considering the position of the acanthocephalan species within Syndermata, it is inferred that obligatory parasitism characteristic of acanthocephalans was acquired after the common ancestor of acanthocephalans diverged from its sister group, Bdelloidea. Additional comparison of complete mtDNA sequences from unsampled acanthocephalan lineages, especially classes Polyacanthocephala and Eoacanthocephala, is required to test if mtDNA provides reliable information for the evolutionary relationships and pattern of life history diversification found in the syndermatan groups.     

Comparisons of direct extraction methods of microbial DNA from different paddy soils

Molecular analyses for the study of soil microbial communities often depend on the direct extraction of DNA from soils. The present work compares the effectiveness of three different methods of extracting microbial DNA from seven different paddy soils. Comparison among different DNA extraction methods against different paddy soil samples revealed a marked variation in DNA yields from 3.18–20.17 μg DNA/g of dry soil. However, irrespective of the soil samples and extraction methods the DNA fragment size was >10 kb. Among the methods evaluated, method-C (chemical–enzymatic–mechanical) had better cell lysis efficiency and DNA yield. After purification of crude DNA by Purification Kit, A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ ratios of the DNA obtained by method-C reached up to 2.27 and 1.89, respectively, sustaining the efficacy of this technique in removing humic acid, protein and other contaminants. Results of the comprehensive evaluation of DNA extraction methods suggest that method-C is superior to other two methods (chemical–enzymatic and chemical–mechanical), and was the best choice for extraction of total DNA from soil samples. Since soil type and microbial community characteristics influence DNA recovery, this study provides guidance for choosing appropriate extraction and purification methods according to experimental goals.     

In silico analysis of Single Nucleotide Polymorphisms (SNPs) in human BRAF gene

BRAF gene mutations are frequently seen in both inherited and somatic diseases. However, the harmful mutations for BRAF gene have not been predicted in silico. Owing to the importance of BRAF gene in cell division, differentiation and secretion processes, the functional analysis was carried out to explore the possible association between genetic mutations and phenotypic variations. Genomic analysis of BRAF was initiated with SIFT followed by PolyPhen and SNPs&GO servers to retrieve the 85 deleterious non-synonymous SNPs (nsSNPs) from dbSNP. A total of 5 mutations i.e. c.406T>G (S136A), c.1446G>T (R462I), c.1556 A>G (K499E), c.1860 T>A (V600E) and c.2352 C>T (P764L) that are found to exert benign effects on the BRAF protein structure and function were chosen for further analysis. Protein structural analysis with these amino acid variants was performed by using I-Mutant, FOLD-X, HOPE, NetSurfP, Swiss PDB viewer, Chimera and NOMAD-Ref servers to check their solvent accessibility, molecular dynamics and energy minimization calculations. Our in silico analysis suggested that S136A and P764L variants of BRAF could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks thus explain the functional deviations of protein to some extent. Screening for BRAF, S136A and P764Lvariants may be useful for disease molecular diagnosis and also to design the molecular inhibitors of BRAF pathways.