Ravi Kant

IsoSeq analysis and functional annotation of the infratentorial ependymoma tumor tissue on PacBio RSII platform.

Here, we sequenced and functionally annotated the long reads (1–2 kb) cDNAs library of an infratentorial ependymoma tumor tissue on PacBio RSII by Iso-Seq protocol using SMRT technology. 577 MB, data was generated from the brain tissues of ependymoma tumor patient, producing 1,19,313 high-quality reads assembled into 19,878 contigs using Celera assembler followed by Quiver pipelines, which produced 2952 unique protein accessions in the nr protein database and 307 KEGG pathways. Additionally, when we compared GO terms of second and third level with alternative splicing data obtained through HTA Array2.0. We identified four and twelve transcript cluster IDs in Level-2 and Level-3 scores respectively with alternative splicing index predicting mainly the major pathways of hallmarks of cancer. Out of these transcript cluster IDs only transcript cluster IDs of gene PNMT, SNN and LAMB1 showed Reads Per Kilobase of exon model per Million mapped reads (RPKM) values at gene-level expression (GE) and transcript-level (TE) track. Most importantly, brain-specific genes–—PNMT, SNN and LAMB1 show their involvement in Ependymoma.

Genotype 3b of human parvovirus B19 detected from hospitalized children with solid malignancies in a North Indian tertiary care hospital

Human parvovirus B19 (B19V) infection is known to cause serious consequences in immuno‐compromized individuals. The present cross sectional study was designed to estimate the prevalence and genotype distribution of B19V in children receiving chemotherapy for solid malignancies at a tertiary care hospital in North India during October 2013 to May 2015. Serum samples from all the patients were tested for anti‐B19V IgM and IgG antibodies and for B19V‐DNA as soon as received. Samples testing positive for B19V‐DNA were subjected to viral load estimation and to genotype determination by sequencing. Total 96 children were enrolled of which 9 (9.3%), 32 (33.3%), and 25 (26%) tested positive for anti‐B19V IgM, anti‐B19V IgG, and B19V‐DNA, respectively. The viral load of B19V‐DNA positive children ranged from 5.5 × 102 to 3.5 × 1012 copies/ml. Accordingly children were divided into three groups: group I, with acute infection (n = 25); group II, previously exposed (n = 27), and group III, negative for B19V infection or with inappropriate antibody response (n = 44). B19V positivity was significantly associated (P‐value < 0.0001) with a history of blood transfusion in the past 6 months, severe anemia (hemoglobin levels <6 gm%) and thrombocytopenia (platelets <150,000/cu.mm.). Sequence analysis of 21 of 25 DNA positive samples showed that all of them were Genotype 3b that clustered into three groups. All the sequences within each cluster were identical. The nucleotide identity of the sequences suggests a nosocomial outbreak of B19V during the study period. Children on chemotherapy for solid tumors should be routinely screened for B19V infection by both serology and PCR. J. Med. Virol. 88:1922–1929, 2016.

Mesenchymal stem cells in regenerative medicine: a new paradigm for degenerative bone diseases

The mesenchymal stem cells (MSCs) have been the epicenter of regenerative medicine since their identification in the 1970s, due to their ability to differentiate into various cell types, their immunosuppressive function, and their ability to home to injury sites. Initially, MSCs were discovered from bone marrow as adherent cells that have the potential to differentiate into bone cells [1–3]. Since inception from bone marrow, analogous cells have been successfully isolated from various sources. Since then, MSCs from bone marrow have been used as a positive control for MSCs isolated from various tissues. A functional characteristic of MSCs is their ability to differentiate into ectoderm, mesoderm and endoderm tissues including bone, neurons, muscles, hepatocytes, skin, among others. MSCs may be characterized based on a panel of surface markers, distinguishing them from endothelial, hematopoietic and monocyte cells. MSCs are typically positive for CD44, CD73, CD90 (Thy-1) and CD105 (endoglin), and negative for hematopoietic (CD45 and TER119 markers) and endothelial (CD31, von Willebrand factor) markers [2,4]. A recent advancement in this field is the isolation of MSCs from dental origin including dental pulp, periodontal ligaments, apical papilla, among others; and we are achieving some positive outcomes with MSCs from dental sources [ Singh SK, Saxena SK, Pers. Comm. ]. Recently, it has been discovered that MSCs could be isolated from nasal polyp tissues, thus providing a potential new source of multipotent MSCs [5]. According to the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy, the minimal criteria of standardization of human MSCs are: first, MSCs must be adherent to the surface of standard plastic culture vessels; second, MSCs must express CD105, CD73, CD90 and low levels of MHC-I and be negative for MHCII, CD45, CD34, CD14 or CD11b surface molecules; and third, MSCs must be able to differentiate into osteoblasts, adipocytes and chondroblasts in vitro [4].

Multiomics approach showing genome-wide copy number alterations and differential gene expression in different types of North-Indian pediatric brain tumors

PURPOSE Based on copy number alterations and transcriptional profiles, the posterior fossa tumors (medulloblastoma (MB), ependymoma and pilocytic astrocytoma) have been classified into various subgroups. The study design was aimed to identify and catalog genome-wide copy number alterations and differential gene expression in different types of North-Indian pediatric posterior fossa tumors and matched control tissue through Molecular Inversion Probe (MIP) Based and Human Transcriptome Array. EXPERIMENTAL DESIGN MIP based OncoScan Array and Human Transcriptome Array 2.0 were used to molecularly-categorize histopathologically and immunohistochemically proven tumor samples on the basis of copy number variations and altered gene expression patterns and/or alternative splicing events. RESULTS Based on molecular, histopathological/immunohistochemical and age-dependent factors MB was subgrouped into group-3 MB, Wnt and SHH; ependymoma into balanced, numerical and structural/anaplastic; and pilocytic astrocytoma was stratified age-dependently. Compared with the vermis tissue of MB, the vermis tissue of ependymoma showed higher levels of gain and losses compared with their counter tumor parts implicating metastasis within the confined region. Group-3 MB and anaplastic ependymoma represented highest differentially expressed genes both at gene and exon levels in the CN altered regions compared with other subgroups of MB and ependymoma respectively. CONCLUSION This multiomics approach based molecular characterization of posterior fossa tumors together with clinical and histopathological factors may help us in the area of personalized medicine.

Incidence and progression of Parvovirus B19 infection and molecular changes in circulating B19V strains in children with haematological malignancy: A follow up study

The present study was planned to estimate the incidence of human Parvovirus B19 infection and understand its progression in children suffering with hematological malignancy. The circulating B19V genotypes and viral mutations occurring in strains of B19V over one-year period were also studied. Children with malignancies were enrolled consecutively and were followed up for one-year period. Serum sample was collected at the time of enrolment and each follow up visit and was tested for anti B19V IgG and IgM as well as for B19V DNA. At least one B19V DNA positive sample from each patient was processed for sequencing. For patients positive for B19V DNA >1 time and at least 6 months apart, last positive sample from the same patient was also sequenced to study the nucleotide change over time. We have found very high incidence of B19V infection (100%) in the study population. All the patients tested positive for at least one B19V infection parameter (either antibodies or DNA) at least once, over one year of follow up. Cumulative percent positivity of anti B19V IgG, anti B19V IgM and B19V DNA was 85.3%, 45.2% and 72.1% respectively. Genotype 3b was reported, with occasional nucleotide change over one year period. DNA clearance was delayed in spite of appearance of IgG antibodies. Appearance of IgM class of antibodies was either delayed or absent. To conclude, children with haematological malignancies have high incidence of B19V infection with late and short lived serological response and persistence of DNA for long duration.

Prevalence of Parvovirus B19V in Hematological Malignancies and Chronic Anemia

To the Editor: The present study was planned to estimate the prevalence of human Parvovirus B19 (B19V) infection in children suffering with hematological malignancies or chronic anemia. Such hospitalized children (<15 y) were enrolled consecutively from 2011 through 2014. King George’s Medical University’s institutional ethical clearance was obtained (no.52 E.C.M.IIA/P6). Anti B19V IgG and IgM antibodies (ELISA kits by NovaTech Immunodiagnostica, Dietzenbach, Germany) and B19V DNA (by real time PCR [1]) were estimated in serum samples of enrolled patients. Relevant laboratory parameters were noted from hospital records of cases. Statistical analysis was done using GraphPad Prism Version 5. Total 379 children were enrolled [Leukemia: 270 cases, chronic unexplained anemia: 109 cases; boys: 264(69.7%), girls: 115(30.3%)]. Mean age ± SD was 6.89 ± 4.17. A high prevalence (70.2%) of B19V infection (presence of any of the three markers) was seen in the study population. No statistically significant difference was seen in B19V positivity among patients of both disease groups. The anti B19V IgG positivity and total positivity of all the three markers of B19V infection increased but B19V DNA positivity decreased significantly with increasing age (Table 1). Data for hemoglobin levels, total leucocyte count, platelet count and bilirubin levels were available for only 186, 177, 173 and 132 cases respectively. The p values (95% CI) for severe anemia (Hemoglobin level < 8.0 g/dl), leucopenia (TLC < 4000/mm), thrombocytopenia (platelets <1.5 lac/mm) and jaundice (bilirubin level > 1.0 mg/dl) in cases with and without B19V infection were 0.516 (0.87–1.36), 0.17 (0.81– 3.27) , 0 .43 (0 .89–1.37) and 0.51 (0 .84–1.49) respectively. The present study describes high prevalence of B19V infection in immunocompromised children; also shown by earlier studies, varying from 34 to 45% [2–4]. Anti B19V IgG positivity increases with age; an observation reported earlier in healthy individuals [5]. The limitation of the present study was that we could not estimate B19V prevalence in age and sex matched healthy controls due to logistic reasons. We have however estimated earlier, B19V prevalence in blood donors, which indicated frequent B19V exposure in the Indian population [6]. B19V infection was not associated with anemia, thrombocytopenia, leucopenia or jaundice in contrast to earlier studies [7], probably because the patients had underlying diseases where these symptoms are features of disease per se. This indicates that B19V infection should be tested for in all hematological diseases patients irrespective of laboratory parameters, for appropriate patient management. To conclude, B19V infection is common in children with hematological diseases, with frequency increasing with age. Therefore, every such child should be evaluated for B19V infection even in the absence of its specific indications. * Amita Jain amita602002@yahoo.com

Insights into the immunopathogenesis during Japanese encephalitis virus infection

Introduction Encephalitis is an acute inflammation of brain caused by either host immune response or viral infection. According to the World Health Organization (WHO), viral encephalitis is predominantly caused by Japanese encephalitis virus (JEV) which belongs to genus Flavivirus and family Flaviviridae. JEV is the main cause of viral encephalitis in many countries of Asia with an estimated 68,000 clinical cases every year[1,2]. JEV is a positive sense single strand (+ss) RNA virus of about 11 kb. RNA encodes a poly-protein precursor of approximately 3400 amino acid. Host cell signalases and viral encodes proteases would accomplish the processing of poly-protein and give rise to three structural proteins that includes core (C), preMembrane (prM) and envelop (E) and seven non-structural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 which are responsible for the replication, viral transcription and also involves in alteration of host immune responses[3,4]. Interestingly, a larger NS1-related protein NS1’ (NS1 prime) is produced as a result of -1 ribosomal 1Department of Stem Cell / Cell Culture, Centre for Advance Research (CFAR), King George’s Medical University (KGMU), Lucknow 226003 (UP), India 2CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007 (TS), India.

Establishing a deceased donor program in north Indian region: lessons learnt

Living‐related donors are the source of almost all organ transplants in India. However, these donations fall far short of current needs, and there remains a huge disparity between demand and supply of organs. In the last five yr, a consistent increase in deceased donor transplant activity has been observed in some southern Indian states. This report describes our experience of establishing a new deceased donor program in the state of Uttar Pradesh in north India.