Aditya Narayan Sarangi

In silico CD4+ T-cell epitope prediction and HLA distribution analysis for the potential proteins of Neisseria meningitidis Serogroup B--a clue for vaccine development.

Neisseria meningitidis, an exclusive human pathogen, is a major cause of mortality due to meningococcal meningitis and sepsis in many developing countries. Three meningococcal serogroup B proteins, i.e. T-cell stimulating protein A (TspA), autotransporter A (AutA), and IgA-specific serine endopeptidase (IGA1) elicits CD4+ T-cell response and may enhance the effectiveness of meningococcal vaccines by acting as protective immunogens. A very limited data on T-helper cell epitopes in MenB proteins is available. Hence, in silico prediction of peptide sequences which may act as helper T lymphocyte epitopes in MenB proteins was carried out by NetMHCIIpan web server. HLA distribution analysis was done by using the population coverage tool of Immune Epitope Database to determine the fraction of individuals in various populations expected to respond to a given set of predicted T-cell epitopes based on HLA genotype frequencies. Six epitopic core sequences, two from each MenB proteins, i.e. AutA, TspA and IgA1 protease were predicted to associate with a large number of HLA-DR alleles. These six peptides may act as T-cell epitope in more than 95% of populations in 8 out of 12 populations considered. The T-cell stimulation potential of these predicted peptides containing the core epitopic sequences is to be validated by using laboratory experiments for their efficient use as peptide vaccine candidates against N. meningitidis serogroup B.

Prediction of Essential Proteins in Prokaryotes by Incorporating Various Physico-chemical Features into the General form of Chou’s Pseudo Amino Acid Composition

Prediction of essential proteins of a pathogenic organism is the key for the potential drug target identification, because inhibition of these would be fatal for the pathogen. Identification of these proteins requires the use of complex experimental techniques which are quite expensive and time consuming. We implemented Support Vector Machine algorithm to develop a classifier model for in silico prediction of prokaryotic essential proteins based on the physico-chemical properties of the amino acid sequences. This classifier was designed based on a set of 10 physico-chemical descriptor vectors (DVs) and 4 hybrid DVs calculated from amino acid sequences using PROFEAT and PseAAC servers. The classifier was trained using data sets consisting of 500 known essential and 500 non-essential proteins (n=1,000) and evaluated using an external validation set consisting of 3,462 essential proteins and 5,538 non-essential proteins (n=9,000). The performances of individual DV sets were evaluated. DV set 13, which is the combination of composition, transition and distribution descriptor set and hybrid autocorrelation descriptor set, provided accuracy of 91.2% in 10-fold cross-validation of the training set and an accuracy of 89.7% in external validation set and of 91.8% and 88.1% using a different yeast protein dataset. Our result indicates that this classification model can be used for identification of novel prokaryotic essential proteins.

Gut microbiome in children with enthesitis‐related arthritis in a developing country, and the effect of probiotic administration

In Asia, enthesitis‐related arthritis (ERA) is the most frequent category of juvenile idiopathic arthritis. ERA has a strong association with human leucocyte antigen (HLA)‐B27 and subclinical gut inflammation. In an HLA‐B27 transgenic rat model, the presence of Bacteroides bacteria in the gut appears to cause spondyloarthropathy (SpA). Thus, we studied gut microbiota in children with ERA. Stool specimens from 33 patients with ERA and 14 age‐matched healthy controls were studied; none had any gastrointestinal symptom, or had received a drug known to affect gut motility or microbiota in the preceding 6 weeks. From each specimen, a cDNA library for the V3 region of bacterial 16S rRNA was subjected to high‐throughput, massively parallel sequencing. Relationship of the specimens was studied using principal co‐ordinate analysis (PCoA), and abundances of various bacterial taxa and alpha diversity were compared between groups. In eight patients, a repeat faecal specimen was studied after 12 weeks of probiotic therapy. The 55 specimens yielded a median (range) of 397 315 (102 093–1 502 380) high‐quality reads each. In PCoA, gut microbiota from ERA showed a wider dispersion than those from controls. In patients, families Bacteroidaceae and Enterobacteriaceae were more abundant and Prevotellaceae were less abundant than in controls. Also, genera Bacteroides, Entercoccus and Klebsiella were over‐represented and genus Prevotella was under‐represented in ERA patients. Probiotic therapy led to a non‐significant increase in Prevotellaceae. Patients with ERA have a dysbiosis in the gut, with increased abundance of Bacteroides and reduction of Prevotella. Probiotic supplementation in a subset of patients did not reverse these changes significantly.

Proteome mining for drug target identification in Listeria monocytogenes strain EGD-e and structure-based virtual screening of a candidate drug target penicillin binding protein 4.

We used a combination of in-silico approaches to identify 168 promising drug targets in the proteome of a multidrug-resistant Listeria monocytogenes strain; of these, one (PBP4) was particularly promising. Virtual screening using it, followed by reverse docking, revealed four compounds namely NCI524121, CAP332797, NCI524136 and ZINC00518462 as good multi-target leads.

Autoimmune polyendocrine syndrome type 1 in an Indian cohort: a longitudinal study

Objective Autoimmune polyendocrine syndrome type 1 (APS1) is a rare autosomal recessive disorder characterized by progressive organ-specific autoimmunity. There is scant information on APS1 in ethnic groups other than European Caucasians. We studied clinical aspects and autoimmune regulator (AIRE) gene mutations in a cohort of Indian APS1 patients. Design Twenty-three patients (19 families) from six referral centres in India, diagnosed between 1996 and 2016, were followed for [median (range)] 4 (0.2–19) years. Methods Clinical features, mortality, organ-specific autoantibodies and AIRE gene mutations were studied. Results Patients varied widely in their age of presentation [3.5 (0.1–17) years] and number of clinical manifestations [5 (2–11)]. Despite genetic heterogeneity, the frequencies of the major APS1 components (mucocutaneous candidiasis: 96%; hypoparathyroidism: 91%; primary adrenal insufficiency: 55%) were similar to reports in European series. In contrast, primary hypothyroidism (23%) occurred more frequently and at an early age, while kerato-conjunctivitis, urticarial rash and autoimmune hepatitis were uncommon (9% each). Six (26%) patients died at a young age [5.8 (3–23) years] due to septicaemia, hepatic failure and adrenal/hypocalcaemic crisis from non-compliance/unexplained cause. Interferon-α and/or interleukin-22 antibodies were elevated in all 19 patients tested, including an asymptomatic infant. Eleven AIRE mutations were detected, the most common being p.C322fsX372 (haplotype frequency 37%). Four mutations were novel, while six others were previously described in European Caucasians. Conclusions Indian APS1 patients exhibited considerable genetic heterogeneity and had highly variable clinical features. While the frequency of major manifestations was similar to that of European Caucasians, other features showed significant differences. A high mortality at a young age was observed.

Identification of autoimmune polyendocrine syndrome type 1 in patients with isolated hypoparathyroidism.

The prevalence of autoimmune polyendocrine syndrome type 1 (APS1) among isolated hypoparathyroidism (HP) or primary adrenal insufficiency (PAI) is not well established. We studied the frequency of APS1 in patients with HP or PAI by measuring interferon‐α (IFN‐α) antibody levels, a highly sensitive and specific marker for APS1.

Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia

Background Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India. Methods DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1. Results Of 71 subjects with UH (68 male; median age [range], 26 [16–63] years; serum bilirubin 56 [26–219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein. Conclusion Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA promoter alleles.

CRELD1 gene variants and atrioventricular septal defects in Down syndrome

Congenital heart defects (CHD) are seen in around 40% of the Down syndrome patients. Atrioventricular Septal Defect (AVSD) or endocardial cushion defect is commonest form of CHD in these children. CRELD1 gene is implicated in causation of sporadic AVSD. In the present study, we evaluated the association and significance of CRELD1 variants with AVSD in Down syndrome (DS) patients. Sequencing was done in blood samples from 3 groups: group I (DS with AVSD), group II (DS without AVSD) and group III (non-syndromic AVSD cases). Twenty two variants in CRELD1 gene were identified, comprising of sixteen novel and six previously reported variants. However, on the basis of sequence, as well as structure analysis, the variant c.973G>A(p.Glu325Lys) variant was identified only in DS having AVSD group which was predicted to have significant effects on calcium binding of putative CRELD1 protein. Since CRELD1 gene acts as a regulator of calcineurin/NFATc1 signaling which is crucial for the regulation of cardiac development by dephosphorylation of the transcription factor, NFAT(nuclear factor of activated T cells),in cytoplasm, the variation in cb-EGF-like calcium binding domain in CRELD1 protein is likely to have pathogenic consequences. Thus, we conclude that the CRELD1 gene is likely to have a major role in causation of AVSD phenotype in selected DS patients.

Methods for studying gut microbiota: A primer for physicians

Human gastrointestinal tract contains a large variety of microbes, in particular bacteria. Studies in recent years have strongly suggested a role for these microbes, collectively referred to as gut microbiota, in the maintenance of homeostasis during health. In addition, alterations in gut microbiota have been reported in several diseases, including those related to the gastrointestinal tract and several systemic conditions, and are believed to play a pathogenetic role in at least some of these. Given the close association between the human gut and liver, the association with gut microbiota appears to be particularly strong for a wide variety of liver diseases. This piece, aimed primarily at physicians, reviews in brief the methods used to study gut microbiota, with particular emphasis on those that use sequences of bacterial 16S rRNA gene or its components.

Faecal bacterial microbiota in patients with cirrhosis and the effect of lactulose administration

BackgroundGut microbiota may be altered in patients with cirrhosis, and may further change after administration of lactulose. We studied the composition of gut microbiota in patients with cirrhosis and assessed the effect on it of lactulose administration.MethodsStool specimens were collected from 35 patients with cirrhosis (male 26; median [range] age: 42 [29–65] years) and 18 healthy controls (male 14; 44.5 [24–67] years); 21 patients provided another specimen after lactulose administration for 55 [42–77] days. For each, a DNA library of V3 region of bacterial 16S ribosomal RNA was subjected to paired-end Illumina sequencing. Inter-specimen relationship was studied using principal co-ordinate analysis. Abundances of various bacterial taxa, and indices of alpha and beta diversity were compared, between patients and controls, and between specimens collected before and after lactulose.ResultsGut microbiota from cirrhosis patients and controls showed differential clustering, and microbiota from patients with cirrhosis had less marked alpha diversity. Abundances of dominant phyla (Bacteroidetes, Firmicutes and Proteobacteria) were similar. However, patients with cirrhosis had lower abundances of five phyla, namely Tenericutes, Cyanobacteria, Spirochaetes, Elusimicrobia and Lentisphaerae, and differences in abundances of several families and genera than in controls. Lactulose administration did not lead to any change in alpha and beta diversities, species richness and abundances of various bacterial taxa in gut microbiota.ConclusionsGut microbiota in cirrhosis differ from healthy persons and do not change following lactulose administration. The latter suggests that the effect of lactulose on hepatic encephalopathy may not be related to alteration in gut microbiota.