Low level of BRCA2 in peripheral blood lymphocytes is associated with an increased risk for head and neck squamous cell carcinoma (HNSCC) in a population of North-East India: a case-control study.

Background: We investigated the role of DNA repair proteins breast cancer susceptibility 2 (BRCA2), xeroderma pigmentosum group D (XPD) and apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) in determining the risk for head and neck squamous cell cancer (HNSCC) in a case-control study from North-East India. Methods: Expression of BRCA2, XPD and APE1 genes in the matched tumour, normal adjacent tissue (NAT) and blood of 12 HNSCC patients and blood of 8 age- and gender-matched controls was determined by quantitative real-time PCR. Results were validated by expression analysis of the corresponding proteins in the peripheral blood lymphocytes (PBLs) of 228 subjects (106 patients and 122 controls) by a slot-blot immunoassay. Findings: Expression of the BRCA2 and XPD genes in tumour tissue of HNSCC patients declined progressively as the cancer stage advanced, was reverse that of the NAT, but was mirrored by the expression in the blood. BRCA2 and XPD proteins were significantly (p < 0.0001) downregulated in the PBL of HNSCC patients to 71% and 77% the levels in controls, showing significant negative correlation with HNSCC stage (Spearman correlation coefficient (r s) of -0.9060, p < 0.0001 for BRCA2; r s of -0.8008, p < 0.01 for XPD). On the contrary, APE1 was significantly upregulated in PBL of HNSCC patients to 1.47 fold the level in controls, showing significant positive correlation with HNSCC stage (r s of 0.7023, p < 0.01). Classification and regression tree analyses predicted low levels of BRCA2 protein in PBL as the single most important risk factor for HNSCC, independent of gender. Smokers above 36 years of age with low level of BRCA2 appeared to exhibit a 1.78-fold increased risk for HNSCC (with a 1.78-fold increased risk for HNSCC (OR = 1.78, 95% confidence interval (CI) = 0.33-9.52) though this risk was not significant statistically. Similarly, low levels of BRCA2 appeared to indicate a moderate, but non-significant risk for HNSCC in non-smokers aged between 36 and 56 years (OR = 1.15, 95% CI = 0.21-6.37). Conclusions: Low level of BRCA2 protein in the peripheral blood indicates increased risk for HNSCC.

In silico assessment of DNA damage response gene variants associated with head and neck cancer.

Head and neck cancer (HNC), the sixth most common cancer globally, stands first in India, especially Northeast India, where tobacco usage is predominant, which introduces various carcinogens leading to malignancies by accumulating DNA damages. Consequently, the present work aimed to predict the impact of significant germline variants in DNA repair and Tumour Suppressor genes on HNC development. WES in Ion ProtonTM platform on 'discovery set' (n = 15), followed by recurrence assessment of the observed variants on 'confirmation set' (n = 40) using Sanger Sequencing was performed on the HNC-prevalent NE Indian populations. Initially, 53 variants were identified, of which seven HNC-linked DNA damage response gene variants were frequent in the studied populations. Different tools ascertained the biological consequences of these variants, of which the non-coding variants viz. EXO1_rs4150018, RAD52_rs6413436, CHD5_rs2746066, HACE1_rs6918700 showed risk, while FLT3_rs2491227 and BMPR1A_rs7074064 conferred protection against HNC by affecting transcriptional regulation and splicing mechanism. Molecular Dynamics Simulation of the full-length p53 model predicted that the observed coding TP53_rs1042522 variant conferred HNC-risk by altering the structural dynamics of the protein, which displayed difficulty in the transition between active and inactive conformations due to high-energy barrier. Subsequent pathway and gene ontology analysis revealed that EXO1, RAD52 and TP53 variants affected the Double-Strand Break Repair pathway, whereas CHD5 and HACE1 variants inactivated DNA repair cascade, facilitating uncontrolled cell proliferation, impaired apoptosis and malignant transformation. Conversely, FLT3 and BMPR1A variants protected against HNC by controlling tumorigenesis, which requires experimental validation. These findings may serve as prognostic markers for developing preventive measures against HNC.

Genetics of randomly bred cats support the cradle of cat domestication being in the Near East.

Cat domestication likely initiated as a symbiotic relationship between wildcats (Felis silvestris subspecies) and the peoples of developing agrarian societies in the Fertile Crescent. As humans transitioned from hunter-gatherers to farmers ~12,000 years ago, bold wildcats likely capitalized on increased prey density (i.e., rodents). Humans benefited from the cats' predation on these vermin. To refine the site(s) of cat domestication, over 1000 random-bred cats of primarily Eurasian descent were genotyped for single-nucleotide variants and short tandem repeats. The overall cat population structure suggested a single worldwide population with significant isolation by the distance of peripheral subpopulations. The cat population heterozygosity decreased as genetic distance from the proposed cat progenitor's (F.s. lybica) natural habitat increased. Domestic cat origins are focused in the eastern Mediterranean Basin, spreading to nearby islands, and southernly via the Levantine coast into the Nile Valley. Cat population diversity supports the migration patterns of humans and other symbiotic species.

The impact of prehistoric human dispersals on the presence of tobacco-related oral cancer in Northeast India.

BACKGROUND: Northeast (NE) India is a subject of debate for predicting its involvement in prehistoric anatomically modern human (AMH) dispersal. The unique lifestyle and genetic characteristics of native ethnic groups in this region are believed to be responsible for their susceptibility to tobacco-related oral cancer (TrOC). The present study assessed mitochondrial macro-haplogroup (mHG) diversity and TrOC susceptibility autosomal loci to evaluate the impact of prehistoric AMH dispersal on the present day's high TrOC prevalence in major NE Indian ethnics. METHODS: We considered 175 unrelated individuals from 35 ethnic groups and previously published 374 sequences for sequencing-based assessment of mtDNA-based marker by subsequent analyses like haplogroup diversity, phylogenetic, genetic structure by AMOVA, and MDS, descriptive statistics of demographic parameters, and migration analysis. Besides, we selected prolonged tobacco-chewing 124 case-control individuals from similar ethnic backgrounds for genotyping 115 autosomal loci in Sequenom iPLEX MassARRAY™ platform and mined 1000genome data (n = 398) for consequent global admixture and ancestry-specific allele frequencies-based analyses. RESULTS: Our mtDNA-based findings suggested that NE populations were distinct from other Indian populations, owing to the first wave of migration from ancient southern China (∼54kya) and two successive spatial expansion events at âˆ¼45kya and âˆ¼43kya. Consequently, it probably acted as another source for prehistoric AMH dispersal in N/NE Asia. Besides, the second wave of back-migration from SE Asia (∼40kya) probably replaced the mitochondrial footprints of survivors from the first migrants and introduced the TrOC susceptibility traits in this region. Afterward, the autosomal marker-based observations on the transition of the disease-associated admixture component 'K6' from SE Asia reconfirmed these results. Moreover, we also observed that the mitochondrial mHG 'R' is significantly associated with the risk of TrOC (OR > 9.5) in NE India. Furthermore, the possible onset of the phenotypic expression of those traits was predicted at âˆ¼4kya, thus, contributing to present-day's TrOC prevalence. CONCLUSIONS: This study reflects its uniqueness by revealing an updated AMH dispersal route for the peopling in and out of NE India, which probably introduced the disease-causing traits in the ancestral NE Indian population. Those traits were then imprinted in their genome to get transferred through their respective generations, forming the present-day's TrOC-prevalent NE Indian population.

Drug repurposing and relabeling for cancer therapy: Emerging benzimidazole antihelminthics with potent anticancer effects.

Origin of drug and radio-refractory clones, cancer stem-like cells, and rapid angiogenesis and metastasis are among the primary concerns that limit the efficacy of anticancer treatments, emphasizing the urgency of developing new therapeutics. Factors like high attrition rates, huge investments, patients' heterogeneity, and diverse molecular subtypes have challenged the rapid development of anticancer drugs. Treatment with repurposing pleiotropic benzimidazole antihelminthics, like mebendazole, albendazole, and flubendazole has recently opened a new window, owing to their easy access, low cost as a generic drug, and long track record of safe use in the human population. This review highlights the outcomes of preclinical and clinical studies of these drugs as a potent anticancer agent(s) conducted in the last two decades. Substantial preclinical studies, as well as limited clinical trials, suggest noteworthy anticancer potency of these pleiotropic benzimidazoles, particularly as potent microtubule disrupting, anti-angiogenic, and anti-metastatic agents, inhibitors of the immune checkpoint, hypoxia-inducible factor, epithelial-mesenchymal transition, cancer stemness, and multidrug resistance protein 1, and inducers of apoptosis and M1 polarization. These anticancer effects are attributed to multiple action points, including intrinsic apoptosis, canonical Wnt/ß-catenin, JAK/STAT-3, JNK, MEK/ERK, and hedgehog signaling pathways. The effective anticancer properties of mebendazole, albendazole, and flubendazole either alone or synergistically with frontline drugs, warrant their validation through controlled clinical trials to use them as promising avenues to anticancer therapy.

Genetic status of indigenous poultry (red jungle fowl) from India.

The global biodiversity of domesticated red jungle fowl (Gallus gallus) is gradually eroding by replacement with commercial poultry breeds and results loss of valuable genetic and physical traits like resistance to disease, extreme environment, etc. posing a threat to the poultry genetic resources. Very fewer reports exist on Indian poultry diversity, especially native chicken of India. Therefore, species identification and inventorying of the poultry genetic resource is indispensable. Thus, the present study aimed to characterize indigenous chicken from bio-diversity hotspot of Sunderban and Northeast India using DNA sequence based barcoding approach. A total of 15 CO1 (Cytochrome c Oxidase-I) DNA barcode of different indigenous chicken were newly sequenced along with 6 previously published sequences from our laboratory and compared with the available data of distinctive genera of Phasianidae as per the standard protocol and are identified as Gallus gallus. About 98.96% of the Phasianid birds were successfully delimitated into the respective species except for 12 congeneric pairs whose minimum interspecific K2P (Kimura 2-parameter) distance overlaps with the maximum intraspecific distance (3.9%). The least genetic divergence is observed between G. gallus and G. varius (0.013%) and highest between G. gallus and G. lafayettei (0.059%). The NJ tree showed a cohesive clustering of indigenous chicken with G. gallus and distinct with respect to all the different species under study, thereby revealing their taxonomic position except for few G. sonneratti that showed mixed clustering with G. gallus. This may be due to the genetic introgression between the species. Nevertheless, the study for the first time provided the molecular identification tag of indigenous poultry from biodiversity hotspot of East and Northeast India and will remain as a potential guide to recognize inimitable and valuable poultry genetic resources for future needs.

The GSTM1 and GSTT1 Null Genotypes Increase the Risk for Type 2 Diabetes Mellitus and the Subsequent Development of Diabetic Complications: A Meta-analysis.

BACKGROUND: Studies pertaining to association of GSTM1 and GSTT1 null genotypes with risk of T2DM and its complications were often inconclusive, thus spurring the present study. METHODS: Meta-analysis of 25 studies for evaluating the role of GSTM1/GSTT1 null polymorphisms in determining the risk for T2DM and 17 studies for evaluating the role of GSTM1/GSTT1 null polymorphisms in development of T2DM related complications were conducted. RESULTS: Our study revealed an association between GSTM1 and GSTT1 null polymorphism with T2DM (GSTM1; OR=1.37;95% CI =1.10-1.70 and GSTT1; OR=1.29;95% CI =1.04-1.61) with an amplified risk of 2.02 fold for combined GSTM1-GSTT1 null genotypes. Furthermore, the GSTT1 null (OR=1.56;95%CI=1.38-1.77) and combined GSTM1-GSTT1 null genotypes (OR=1.91;95%CI=1.25- 2.94) increased the risk for development of T2DM related complications, but not the GSTM1 null genotype. Stratified analyses based on ethnicity revealed GSTM1 and GSTT1 null genotypes increase the risk for T2DM in both Caucasians and Asians, with Asians showing much higher risk of T2DM complications than Caucasians for the same. DISCUSSION: GSTM1, GSTT1 and combined GSTM1-GSTT1 null polymorphism may be associated with increased risk for T2DM; while GSTT1 and combined GSTM1-GSTT1 null polymorphism may increase the risk of subsequent development of T2DM complications with Asian population carrying an amplified risk for the polymorphism. CONCLUSION: Thus GSTM1 and GSTT1 null genotypes increases the risk for Type 2 diabetes mellitus alone, in combination or with regards to ethnicity.

Metabolic Phase I (CYPs) and Phase II (GSTs) Gene Polymorphisms and Their Interaction with Environmental Factors in Nasopharyngeal Cancer from the Ethnic Population of Northeast India.

Multiple genetic and environmental factors and their interaction are believed to contribute in the pathogenesis of Nasopharyngeal Cancer (NPC). We investigate the role of Metabolic Phase I (CYPs) and Phase II (GSTs) gene polymorphisms, gene-gene and gene-environmental interaction in modulating the susceptibility to NPC in Northeast India. To determine the association of metabolic gene polymorphisms and environmental habits, 123 cases and 189 controls blood/swab samples were used for PCR and confirmed by Sanger sequencing. Analysis for GSTM1 and GSTT1 gene polymorphism was done by multiplex PCR. The T3801C in the 3'- flanking region of CYP1A1 gene was detected by PCR-RFLP method. The Logistic regression analysis was used to estimate odds ratios (OR) and 95% confidence intervals (95% CI). The GSTM1 null genotype alone (OR = 2.76) was significantly associated with NPC risk (P < 0.0001). The combinations of GSTM1 null and GSTT1 null genotypes also higher, 3.77 fold (P < 0.0001), risk of NPC, while GSTM1 null genotype along with CYP1A1 T3801C TC + CC genotype had 3.22 (P = 0.001) fold risk. The most remarkable risk was seen among individual carrying GSTM1 null, GSTT1 null genotypes and CYP1A1 T3801C TC + CC genotypes (OR = 5.71, P = 0.001). Further; analyses demonstrate an enhanced risk of NPC in smoked meat (OR = 5.56, P < 0.0001) and fermented fish consumers (OR = 5.73, P < 0.0001) carrying GSTM1 null genotype. An elevated risk of NPC was noted in smokers (OR = 12.67, P < 0.0001) and chewers (OR = 5.68, P < 0.0001) with GSTM1 null genotype. However, smokers had the highest risk of NPC among individuals carrying GSTT1 null genotype (OR = 4.46, P = 0.001) or CYP1A1 T3801C TC + CC genotype (OR = 7.13, P < 0.0001). The association of null genotypes and mutations of metabolic neutralizing genes along with the environmental habits (tobacco smokers and chewers, smoke meat, fermented fishes) can be used as a possible biomarker for early detection and preventive measure of NPC.

Association of DFNA5, SYK, and NELL1 variants along with HPV infection in oral cancer among the prolonged tobacco-chewers.

Southeast Asia, especially India, is well known for the highest use of smokeless tobacco. These products are known to induce oral squamous cell carcinoma. However, not all long-term tobacco-chewers develop oral squamous cell carcinoma. In addition, germline variants play a crucial role in susceptibility, prognosis, development, and progression of the disease. These prompted us to study the genetic susceptibility to oral squamous cell carcinoma among the long-term tobacco-chewers. Here, we presented a retrospective study on prolonged tobacco-chewers of Northeast India to identify the potential protective or risk-associated germline variants in tobacco-related oral squamous cell carcinoma along with HPV infection. Targeted re-sequencing (n = 60) of 170 genetic regions from 75 genes was carried out in Ion-PGM™ and validation (n = 116) of the observed variants was done using Sequenom iPLEX MassARRAY™ platform followed by polymerase chain reaction-based HPV genotyping and p16-immunohistochemistry study. Subsequently, estimation of population structure, different statistical and in silico approaches were undertaken. We identified one nonsense-mediated mRNA decay transcript variant in the DFNA5 region (rs2237306), associated with Benzo(a)pyrene, as a protective factor (odds ratio = 0.33; p = 0.009) and four harmful (odds ratio > 2.5; p < 0.05) intronic variants, rs182361, rs290974, and rs169724 in SYK and rs1670661 in NELL1 region, involved in genetic susceptibility to tobacco- and HPV-mediated oral oncogenesis. Among the oral squamous cell carcinoma patients, 12.6% (11/87) were HPV positive, out of which 45.5% (5/11) were HPV16-infected, 27.3% (3/11) were HPV18-infected, and 27.3% (3/11) had an infection of both subtypes. Multifactor dimensionality reduction analysis showed that the interactions among HPV and NELL1 variant rs1670661 with age and gender augmented the risk of both non-tobacco- and tobacco-related oral squamous cell carcinoma, respectively. These suggest that HPV infection may be one of the important risk factors for oral squamous cell carcinoma in this population. Finally, we newly report a DFNA5 variant probably conferring protection via nonsense-mediated mRNA decay pathway against tobacco-related oral squamous cell carcinoma. Thus, the analytical approach used here can be useful in predicting the population-specific significant variants associated with oral squamous cell carcinoma in any heterogeneous population.

Assessment of DNA repair susceptibility genes identified by whole exome sequencing in head and neck cancer.

Head and neck cancer (HNC), the sixth most common cancer globally, stands second in India. In Northeast (NE) India, it is the sixth most common cause of death in males and seventh in females. Prolonged tobacco and alcohol consumption constitute the major etiological factors for HNC development, which induce DNA damage. Therefore, DNA repair pathway is a crucial system in maintaining genomic integrity and preventing carcinogenesis. The present work was aimed to predict the consequence of significant germline variants of the DNA repair genes in disease predisposition. Whole exome sequencing was performed in Ion Proton™ platform on 15 case-control samples from the HNC-prevalent states of Manipur, Mizoram, and Nagaland. Variant annotation was done in Ion Reporter™ as well as wANNOVAR. Subsequent statistical and bioinformatics analysis identified significant exonic and intronic variants associated with HNC. Amongst our observed variants, 78.6% occurred in ExAC, 94% reported in dbSNP and 5.8% & 9.3% variants were present in ClinVar and HGMD, respectively. The total variants were dispersed among 199 genes with DSBR and FA pathway being the most mutated pathways. The allelic association test suggested that the intronic variants in HLTF and RAD52 gene significantly associated (P < 0.05) with the risk (OR > 5), while intronic variants in PARP4, RECQL5, EXO1 and PER1 genes and exonic variant in TDP2 gene showed protection (OR < 1) for HNC. MDR analysis proposed the exonic variants in MSH6, BRCA2, PALB2 and TP53 genes and intronic variant in RECQL5 genetic region working together during certain phase of DNA repair mechanism for HNC causation. In addition, other intronic and 3'UTR variations caused modifications in the transcription factor binding sites and miRNA target sites associated with HNC. Large-scale validation in NE Indian population, in-depth structure prediction and subsequent simulation of our recognized polymorphisms is necessary to identify true causal variants related to HNC.

Detection of p16 Promoter Hypermethylation by Methylation-Specific PCR.

DNA methylation plays a decisive role in the regulation and control of gene expression. DNA methylation is a covalent modification, in which a methyl group is attached to the 5th carbon of the cytosine ring of a CpG dinucleotide that is located upstream from the promoter region of a gene. Promoter hypermethylation (gain of DNA methylation) of the p16 gene may cause silencing of gene expression and plays an important role in cancer. Therefore, detection of the methylation status of p16 gene is an important tool in epigenetic studies of various human cancers. The methylation-specific PCR (MSP) is the most commonly used technique for studying DNA methylation. This technique is based on bisulfite modification of DNA, which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (Cm) unchanged. Here we describe the bisulfite modification of DNA samples and detection of promoter methylation of p16 gene from bisulfite-treated DNA using MSP. In MSP, modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers for the p16 gene separately. The PCR amplified products are then analyzed in a 2.5-3% agarose gel containing ethidium bromide. The PCR amplified band generated by specific sets of primers is used to determine the methylation status of the p16 gene.

Application and optimization of minimally invasive cell-free DNA techniques in oncogenomics.

The conventional method of measuring biomarkers in malignant tissue samples has already given subversive growth in cancer diagnosis, prognosis, and therapy selection. However, the regression and heterogeneity associated with tumor tissue biopsy have urged for the development of an alternative approach. Considering the limitations, cell-free DNA has emerged as a surrogate alternative, facilitating preoperative chemoradiotherapy (p < 0.0001) treatment response in rectal cancer and detection of biomarker in lung cancer. This potential of cell-free DNA in several other cancers has yet to be explored based on clinical relevance by optimizing the preanalytical factors. This review has highlighted the crucial parameters from blood collection to cell-free DNA analysis that has a significant impact on the accuracy and reliability of clinical data. The quantity of cell-free DNA is also a limiting factor. Therefore, a proper preanalytical factor for blood collection, its stability, centrifugation speed, and plasma storage condition are to be optimized for developing cancer-specific biomarkers useful for clinical purpose. Liquid biopsy-based origin of cell-free DNA has revolutionized the area of cancer research. Lack of preanalytical and analytical procedures may be considered for identification of novel biomarkers through next-generation sequencing of tumor-originated cell-free DNA in contradiction to tissue biopsy for cancer-specific biomarkers.

Genetic assessment of leech species from yak (Bos grunniens) in the tract of Northeast India.

Yak is an iconic symbol of Tibet and high altitudes of Northeast India. It is highly cherished for milk, meat, and skin. However, yaks suffer drastic change in milk production, weight loss, etc, when infested by parasites. Among them, infestation by leeches is a serious problem in the Himalayan belt of Northeast India. The parasite feeds on blood externally or from body orifices, like nasopharynx, oral, rectum, etc. But there has been limited data about the leech species infesting the yak in that region because of the difficulties in morphological identification due to plasticity of the body, changes in shape, and surface structure and thus, warrants for the molecular characterization of leech. In anticipation, this study would be influential in proper identification of leech species infesting yak track and also helpful in inventorying of leech species in Northeast India. Here, we investigated, through combined approach of molecular markers and morphological parameters for the identification of leech species infesting yak. The DNA sequences of COI barcode fragment, 18S and 28S rDNA, were analyzed for species identification. The generated sequences were subjected to similarity match in global database and analyzed further through Neighbour-Joining, K2P distance based as well as ML approach. Among the three markers, only COI was successful in delineating species whereas the 18S and 28S failed to delineate the species. Our study confirmed the presence of the species from genus Hirudinaria, Haemadipsa, Whitmania, and one species Myxobdella annandalae, which has not been previously reported from this region.

Cell-free mitochondrial DNA copy number variation in head and neck squamous cell carcinoma: A study of non-invasive biomarker from Northeast India.

Head and neck squamous cell carcinoma is the most commonly diagnosed cancer worldwide. The lifestyle, food habits, and customary practices manifest the Northeast Indian population toward higher susceptibility to develop head and neck squamous cell carcinoma. Here, we have investigated the association of smoke and smokeless tobacco, and alcohol with copy number variation of cell-free mitochondrial DNA and cell-free nuclear DNA in cases and controls. Cell-free DNA from plasma was isolated from 50 head and neck squamous cell carcinoma cases and 50 controls with informed written consent using QIAamp Circulating Nucleic Acid Kit. Real-time polymerase chain reaction was done for copy number variation in cell-free mitochondrial DNA and cell-free nuclear DNA. Receiver operating characteristic curve analysis was performed to evaluate the diagnostic application between the two study groups using clinicopathological parameters. The levels of cell-free nuclear DNA and cell-free mitochondrial DNA of cases in association with smoke and smokeless tobacco, alcohol with smoking (p < 0.05) were significantly higher (p < 0.01 and p < 0.001, respectively) than controls. Using receiver operating characteristic curve analysis between head and neck squamous cell carcinoma cases and controls, we distinguished cell-free mitochondrial DNA (cutoff: 19.84 raw Ct; sensitivity: 84%; specificity: 100%; p < 0.001) and cell-free nuclear DNA (cutoff: 463,282 genomic equivalent/mL; sensitivity: 53%; specificity: 87%; p < 0.001). The copy number variation in cases (cell-free nuclear DNA: 5451.66 genomic equivalent/mL and cell-free mitochondrial DNA: 29,103,476.15 genomic equivalent/mL) and controls (cell-free nuclear DNA: 1650.9 genomic equivalent/mL and cell-free mitochondrial DNA: 9,189,312.54 genomic equivalent/mL), respectively. Our result indicates that the cell-free mitochondrial DNA content is highly associated with smoke and smokeless tobacco, betel quid chewing, and alcohol which shows greater promises, holding the key characteristics of diagnostic biomarkers, that is, minimal invasiveness, high specificity, and sensitivity.

Mini-DNA barcode in identification of the ornamental fish: A case study from Northeast India.

The ornamental fishes were exported under the trade names or generic names, thus creating problems in species identification. In this regard, DNA barcoding could effectively elucidate the actual species status. However, the problem arises if the specimen is having taxonomic disputes, falsified by trade/generic names, etc., On the other hand, barcoding the archival museum specimens would be of greater benefit to address such issues as it would create firm, error-free reference database for rapid identification of any species. This can be achieved only by generating short sequences as DNA from chemically preserved are mostly degraded. Here we aimed to identify a short stretch of informative sites within the full-length barcode segment, capable of delineating diverse group of ornamental fish species, commonly traded from NE India. We analyzed 287 full-length barcode sequences from the major fish orders and compared the interspecific K2P distance with nucleotide substitutions patterns and found a strong correlation of interspecies distance with transversions (0.95, p<0.001). We, therefore, proposed a short stretch of 171bp (transversion rich) segment as mini-barcode. The proposed segment was compared with the full-length barcodes and found to delineate the species effectively. Successful PCR amplification and sequencing of the 171bp segment using designed primers for different orders validated it as mini-barcodes for ornamental fishes. Thus, our findings would be helpful in strengthening the global database with the sequence of archived fish species as well as an effective identification tool of the traded ornamental fish species, as a less time consuming, cost effective field-based application.

Design of character-based DNA barcode motif for species identification: A computational approach and its validation in fishes.

The DNA barcodes are generally interpreted using distance-based and character-based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance-based approach has a limitation in defining a universal species boundary across the taxa as the rate of mtDNA evolution is not constant throughout the taxa. However, character-based approach more accurately defines this using a unique set of nucleotide characters. The character-based analysis of full-length barcode has some inherent limitations, like sequencing of the full-length barcode, use of a sparse-data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154-bp fragment, from the transversion-rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species-specific barcode motifs for 109 species by the character-based method, which successfully identifies the correct species using a pattern-matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species-specific mini-barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini-barcode approach will greatly benefit the field-based system of rapid species identification.

Genetic variants of the DNA repair genes from Exome Aggregation Consortium (EXAC) database: significance in cancer.

DNA repair pathway is a primary defense system that eliminates wide varieties of DNA damage. Any deficiencies in them are likely to cause the chromosomal instability that leads to cell malfunctioning and tumorigenesis. Genetic polymorphisms in DNA repair genes have demonstrated a significant association with cancer risk. Our study attempts to give a glimpse of the overall scenario of the germline polymorphisms in the DNA repair genes by taking into account of the Exome Aggregation Consortium (ExAC) database as well as the Human Gene Mutation Database (HGMD) for evaluating the disease link, particularly in cancer. It has been found that ExAC DNA repair dataset (which consists of 228 DNA repair genes) comprises 30.4% missense, 12.5% dbSNP reported and 3.2% ClinVar significant variants. 27% of all the missense variants has the deleterious SIFT score of 0.00 and 6% variants carrying the most damaging Polyphen-2 score of 1.00, thus affecting the protein structure and function. However, as per HGMD, only a fraction (1.2%) of ExAC DNA repair variants was found to be cancer-related, indicating remaining variants reported in both the databases to be further analyzed. This, in turn, may provide an increased spectrum of the reported cancer linked variants in the DNA repair genes present in ExAC database. Moreover, further in silico functional assay of the identified vital cancer-associated variants, which is essential to get their actual biological significance, may shed some lights in the field of targeted drug development in near future.

A murine model of type 2 diabetes mellitus developed using a combination of high fat diet and multiple low doses of streptozotocin treatment mimics the metabolic characteristics of type 2 diabetes mellitus in humans.

INTRODUCTION: A murine model of type 2 diabetes mellitus was used to compare the antidiabetic effects of the dipeptidyl peptidase-4 (DPP4) inhibitor vildagliptin and biguanide, metformin. METHODS: Swiss albino mice (n=20 males; n=25 females) were given high fat diet (HFD) ad libitum for 3weeks followed by low dose (40mgkg-1 body weight, bw daily) of streptozotocin (STZ) intraperitoneally five times from the 22nd day of treatment onwards, with HFD continued up to 26th day. Controls (n=15 males; n=15 females) were fed normal balanced diet without administration of STZ. Successful induction of diabetes mellitus was confirmed by testing for fasting blood glucose, intraperitoneal glucose tolerance and intraperitoneal insulin sensitivity. Diabetic mice were administered vildagliptin (10mgkg-1 bw daily) and metformin (50mgkg-1 bw daily) orally for 4weeks. Control, diabetic, vildagliptin and metformin-treated diabetic mice were evaluated for alterations in lipid profile using blood serum and histopathology and oxidative stress using tissues including liver, kidney and heart. RESULTS: Diabetic mice showed significant alterations in lipid profile, tissue histopathology, impaired glucose tolerance, lower insulin sensitivity and elevated lipid peroxidation and protein carbonylation, with depressed catalase activity, when compared to age and gender-matched controls. Metformin and vildagliptin ameliorated the abovementioned diabetic conditions, with vildagliptin found to be more effective. DISCUSSION: A murine model developed by the combination of HFD and multiple low dose of STZ mimics the metabolic characteristics of type 2 diabetes mellitus in humans, and may be useful for antidiabetic drug screening.

BRCA1 and MDM2 as independent blood-based biomarkers of head and neck cancer.

We investigated the role of BRCA1, MDM2, and p53 in the pathogenesis of head and neck cancer (HNC) and evaluated their potential utility as blood-based predictive biomarkers of HNC. Immunostaining of tissue biopsies and whole blood lymphocytes (WBL) of 36 HNC patients were evaluated by immunohistochemistry (IHC) and immunocytochemistry (ICC), respectively. The staining intensities of BRCA1 and MDM2 in matched tissue and blood samples were significantly associated with cancer stage. Furthermore, the cellular levels of BRCA1, MDM2, and p53 were evaluated in peripheral blood lymphocytes (PBL) of 134 HNC patients and 126 controls by slot blotting. Expression levels of all three proteins in PBL of HNC patients varied significantly with respect to those of controls (p < 0.0001) with BRCA1 downregulated to 75 % of control and MDM2 and p53 upregulated to 1.7- and 1.4-fold the control level, respectively. Moreover, positive correlation was observed between expression levels of BRCA1, MDM2, and p53 in matched tissue biopsies-WBL (r s = 0.840, 0.754, and 0.806, respectively), tissue biopsies-PBL (r s = 0.745, 0.736, and 0.776, respectively), and PBL-WBL (r s = 0.709, 0.758, and 0.740, respectively), validating the hypothesis that these proteins may serve as blood-based biomarkers of HNC. Bias-corrected and accelerated (BCa) bootstrap cross-validation estimation of receiver operating characteristics (ROC) analysis established BRCA1 (AUC = 0.726, sensitivity = 89 %, NPV = 82 %) and MDM2 (AUC = 0.827, sensitivity = 85 %, NPV = 81 %) as predictive biomarkers for HNC. In conclusion, this study suggests that BRCA1 and MDM2 play a crucial role in the pathogenesis of HNC and could be used independently as predictive biomarkers for HNC.

Polymorphisms of XRCC1 and XRCC2 DNA Repair genes and Interaction with Environmental Factors Influence the Risk of Nasopharyngeal Carcinoma in Northeast India.

Multiple genetic and environmental factors have been reported to play key role in the development of nasopharyngeal carcinoma (NPC). Here, we investigated interactions of XRCC1 Arg399Gln and XRCC2 Arg188His polymorphisms and environmental factors in modulating susceptibility to NPC in Northeast India. One-hundred NPC patients, 90 first-degree relatives of patients and 120 controls were enrolled in the study. XRCC1 Arg399Gln and XRCC2 Arg188His polymorphisms were determined using PCR-RFLP, and the results were confirmed by DNA sequencing. Logistic regression (LR) and multifactor dimensionality reduction (MDR) approaches were applied for statistical analysis. The XRCC1 Gln/Gln genotype showed increased risk (OR=2.76; <0.024) of NPC. However, individuals with both XRCC1 and XRCC2 polymorphic variants had 3.2 fold elevated risk (<0.041). An enhanced risk of NPC was also observed in smoked meat (OR=4.07; P=0.004) and fermented fish consumers (OR=4.34, P=0.001), and tobacco-betel quid chewers (OR=7.00; P=0.0001) carrying XRCC1 polymorphic variants. However, smokers carrying defective XRCC1 gene showed the highest risk (OR = 7.47; <0.0001). On MDR analysis, the best model for NPC risk was the five-factor model combination of XRCC1 variant genotype, fermented fish, smoked meat, smoking and chewing (CVC=10/10; TBA=0.636; <0.0001); whereas in interaction entropy graphs, smoked meat and tobacco chewing showed synergistic interactions with XRCC1. These findings suggest that interaction of genetic and environmental factors might increase susceptibility to NPC in Northeast Indian populations.