Role of HCMV miR-UL70-3p and miR-UL148D in overcoming the cellular apoptosis.

The studies into the pathophysiology of viral miRNAs are still in infancy; the interspecies regulation at the miRNA level fuels the spark of the investigation into the repertoire of virus-host interactions. Reports pertaining to the viral miRNAs role in modulating/evading the host immune response are surging up; we initiated this in silico study to speculate the role of human cytomegalovirus (HCMV)-encoded miRNAs on human antiviral mechanisms such as apoptosis and autophagy. The results indicate that both the above mechanisms were targeted by the HCMV miRNAs, located in the unique long region of the HCMV genome. The proapoptotic genes MOAP1, PHAP, and ERN1 are identified to be the potential targets for the miR-UL70-3p and UL148D, respectively. The ERN1 gene plays a role in the initiation of Endoplasmic reticulum stress-induced apoptosis as well as autophagosome formation. This study shows that HCMV employs its miRNA repertoire for countering the cellular apoptosis and autophagy, particularly the mitochondrial-dependent intrinsic pathway of apoptosis. In addition, the homology studies reveal no HCMV miRNA bears sequence homology with human miRNAs.

The efficacy of diagnostic battery in Pott's disease: A prospective study.

BACKGROUND: The diagnosis of Pott's disease is mostly based on clinicoradiological observations substantiated by the bacterial culture, staining and histopathology. Since, no single technique is enough to conclude Pott's disease in diagnosis, the present study was undertaken to correlate the clinicoradiological, microbiological, histopathological and molecular method to evaluate the effectiveness in diagnosis of Pott's disease. MATERIALS AND METHODS: 62 clinicoradiologically suspected cases of Pott's disease were included in this study. The specimens for diagnostic work up were collected either during surgery or by computed tomography guided fine needle aspiration. All these specimens were tested for tuberculosis (TB) through Ziehl-Neelsen (ZN) microscopy, BACTEC culture, histopathology and polymerase chain reaction (PCR). The final diagnosis was established by the results of performed tests and clinicoradiological improvement of cases at the end of 6 months on anti tubercular treatment. RESULTS: Out of 62 cases, 7 were excluded from this study as these were turned out to be neoplastic lesions on histopathology. Amongst remaining 55 cases, the TB was diagnosed in 39 (71%) on histopathology, 37 (67.5%) on PCR, 27 (49%) on BACTEC culture and 20 (36.3%) on ZN microscopy. Ultimately 45 cases were tested as positive and 10 were detected as negative for TB in combination of ZN microscopy, BACTEC culture and histopathology. PCR was positive in 37 of 45 cases and 10/55 cases remained negative. On clinical analysis of these 10 cases, it was noted that these were cases of relapse/poor compliance. The combination of PCR and histopathology was also shown positive for TB in 45 cases. Hence, the PCR showed a fair positive agreement (Κ(c) = 0.63) against the combined results of all performed traditional methods. CONCLUSIONS: The combination of PCR and histopathology is a rapid and efficient tool for diagnosis of Pott's disease.

KIT proto-oncogene exon 8 deletions at codon 419 are highly frequent in acute myeloid leukaemia with inv(16) in Indian population.

The KIT gene is a receptor tyrosine kinase class III expressed by early hematopoietic progenitor cells and plays a significant role in hematopoietic stem cell proliferation, differentiation and survival which is considered to be a remarkable feature in the course of growth of acute myeloid leukaemia (AML). Owing to insufficient study of mutations in the KIT gene, the diagnosis and rate of recurrence of these mutations with divergent subtypes in AML cases in India is of concern. In order to find out the frequency of mutations of KIT gene exon 8 in 109 AML cases, we have performed polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) followed by DNA sequencing and have identified 24 mutations in exon 8 in 13 cases, including deletions at codon 418 (n = 3), 419 (n = 11) and 420 (n = 5) as well as point mutations at codon 417 (n = 1) and 421 (n = 4). In eleven AML cases, exon 8 deletion and point mutations involved the loss at codon Asp419 immoderately conserved cross species placed in the receptor extracellular domain. Frequency elevation of the KIT proto-oncogene exon 8 deletion and point mutations in AML cases allude a crucial function for this region of the receptor extracellular domain. Thus, we report the incidence of acquired mutations in exon 8, with consistent loss at codon Asp419, in 10.09 % of AML cases in a selected Indian population.

Screening of the c-kit gene missense mutation in invasive ductal carcinoma of breast among north Indian population.

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer deaths among females across the world, accounting for 23 % (1.38 million) of total new cancer cases and 14 % (0.45 million) of the total cancer deaths in 2008. c-kit is expressed in mast cell growth factor, cellular migration, proliferation, melanoblasts, haematopoietic progenitors and germ cells. We have designed our study with aim to explore the c-kit gene mutations in invasive ductal carcinoma (IDC) breast. To ascertain the range of mutations in exon 11, 13 and 17 of c-kit gene in 53 cases of IDC breast, we carried out PCR-SSCP followed by DNA sequencing. The mutation frequency of c-kit gene in exon 11, 13 and 17 were 9.43 % (5/53), 1.88 % (1/53) and 3.77 % (2/53), respectively. During our mutational analysis, we have detected five missense mutations in exon 11 (Pro551Leu, Glu562Val, Leu576Phe, His580Tyr and Phe584Leu), one missense mutation in exon 13 (Ser639Pro) and two missense mutations in exon 17 (Arg796Gly and Asn822Ser). It seems that c-kit mutations might participate in breast cancer pathogenesis and may be utilized as predictive marker, since the loss of c-kit positivity is generally linked with different types of breast cancer. Further molecular studies are necessary to validate the association of c-kit gene mutation in IDC breast pathogenesis.

Methylenetetrahydrofolate reductase C677T genetic polymorphisms and risk of leukaemia among the North Indian population.

BACKGROUND: Leukaemia is a heterogeneous disease in which haematopoietic progenitor cells acquire genetic lesions that lead to a block in differentiation, increased self-renewal, and unregulated proliferation. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR), involved in folate metabolism, plays a crucial role in cells because folate availability is important for DNA integrity. The aim of this case-control study was to evaluate the association of the C677T MTHFR gene polymorphism with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphocytic leukaemia (CLL). MATERIALS AND METHODS: A total of 275 leukaemia cases - including AML (n = 112), ALL (n = 81), CML (n = 43), CLL (n = 39) - and 251 age/sex-matched healthy control individuals participated in this study. MTHFR C677T polymorphisms in the cases and controls were evaluated by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). RESULTS: The average MTHFR 677CC, 677CT, 677TT genotype frequencies of total leukaemia cases were 68.73%, 19.64%, and 11.64% in cases, and 71.71%, 24.30%, and 3.98% in healthy controls, respectively. The average frequency of the MTHFR 677T allele was 21.45% among the cases compared to 16.13% among the controls. CONCLUSIONS: In the present case-control study we have observed a higher frequency of the MTHFR 677TT genotype in cases of leukaemia (AML, ALL, CML and CLL) as compared with controls; this might be due to ethnic and geographic variation. As per our findings, although the frequency of the MTHFR 677T allele is moderately high in AML, ALL and CLL, no statistically significant association was found; on the other hand statistically significant association was found in the context of CML cases.

Identification of the c-kit gene mutations in biopsy tissues of mammary gland carcinoma tumor.

C-kit gene is a transmembrane tyrosine kinase that acts as type III receptor for mast cell growth factor and cellular migration, proliferation, survival of melanoblasts, haematopoietic progenitors and primordial germ cells. Apart from the scant information about the pathologies associated with loss-of-function mutations, few reports have proposed role of the c-kit gene in case of carcinogenesis. Apparently, in breast cancer the involvement of c-kit gene mutations has been considered as a rare phenomenon. Thus, we designed our study with aim to investigate the c-kit gene mutation in breast cancer, and their correlation with clinico-pathological findings. We performed mutational analysis of the c-kit gene in 58 cases of malignant breast cancer. With the aim to ascertain the variety of mutations at exon 8, 9, 11, 13, 15 and 17 of c-kit gene in breast cancer, we have done PCR-SSCP followed by DNA sequencing. In breast cancer the c-kit gene mutation rates were 3.44% (02/58) in exon 8, 5.17% (3/58) in exon 9, 5.17% (3/58) in exon 11, 3.44% (2/580 in exon 13, 3.44% (2/58) in exon 15 and 5.17% (3/58) in exon 17, respectively. The overall c-kit mutation frequency in exons 8, 9, 11, 13, 15 and 17 was determined to be 25.86% (15/58). Our study indicates to specify the role of c-kit proto-oncogene mutation in breast cancer. The result signifies that c-kit gene plays a poor role in prognosis of ductal and lobular carcinoma.

The Frequency of Mutations in Exon 11 of the c-kit Gene in Patients With Leukemia.

OBJECTIVE: To determine the frequency of mutations in exon 11 of the c-kit gene in patients with leukemia. MATERIAL AND METHODS: The study included 50 leukemia patients (31 with acute myeloid leukemia, 5 with acutelymphoblastic leukemia, 9 with chronic myeloid leukemia, and 5 with chronic lymphocytic leukemia) that underwentPCR-SSCP, followed by direct DNA sequencing. RESULTS: In all, 28 of the leukemia patients were male and 22 were female, with a mean age of 31.88 years (range: 2-65years). In total, 20 mutations in 19 patients were identified, including Lys550Asn, Tyr568Ser, Ile571Thr, Thr574Pro,Gln575His, Tyr578Pro, Asp579His, His580Gln, Arg586Thr, Asn587Asp, and Arg588Met, as well as novel point mutationsat codons Ile563Lys, Val569Leu, Tyr570Ser, and Pro577Ser. Ile571Leu substitution was observed in 2 patients andTrp582Ser substitution was observed in 3 patients. CONCLUSION: The results suggest that mutations in exon 11 of the c-kit gene might be useful as molecular geneticmarkers for leukemia.

Cellular oncomiR orthologue in EBV oncogenesis.

MicroRNAs are small non-coding RNAs that regulate gene expression at multiple levels. The discovery of virally encoded miRNAs attracted immense attention towards their role in viral replication and pathogenesis. Kaposi's-sarcoma-associated herpes virus encodes miRNA that functions as an orthologue of human cellular miRNA, i.e., hsa-miR-155. Keeping the same view we extended the miRNA-homology search between the miRNAs of humans and Epstein-Barr virus. The In silico analyses shows that EBV encoded miR-BART-5 has a significant 'seed' sequence homology to hsa-miR-18 of humans. Further, the mRNA transcripts of the human genes involved in cellular growth could potentially be targeted by both viral as well as human miRNAs. The known etiological role of hsa-miR-18 as an oncomiR suggests that miR-BART-5 may function as viral oncomiR as observed in EBV-positive gastric carcinoma patients.

Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search.

Use of siRNA is a powerful methodology to particularly knockdown the targeted genes in a sequence specific manner. The potential of siRNA can be harnessed for silencing specific geminiviral genes in papaya and tomato plant hosts, thus making them resistant to the respective viruses. The challenge is in designing exogenous siRNA which can trigger silencing of viral genes irrespective of the genetic variability in different viral isolates and at the same time the selected siRNA does not target any plant gene (off target silencing). In this study, we have designed siRNA from the most conserved regions of viral coat protein (AV1) and replicase (AC1) genes retrieved from different isolates of geminiviruses infecting papaya (PLCV), and tomato (TLCV & TLCV, Northern India), so as to give a broad spectrum resistance and efficient silencing as it is highly homology-dependent strategy. Software siRNA finder (Ambion) was used on the selected conserved sequences in order to select only those putative siRNA oligonucleotides which fulfill all the basic criteria required as per the algorithm. Finally, a cross search using BLAST was performed to confirm that the designed siRNAs do not have any homology to plant genome sequences. The putative siRNA sequences thus designed to target essential genes of geminiviruses and introduced into the plants may facilitate developing papaya and tomato crops with generic resistance to geminiviruses.