Comprehensive Characterization of Stage IIIA Non-Small Cell Lung Carcinoma.

INTRODUCTION: Heterogeneity of non-small cell lung carcinoma (NSCLC) among patients is currently not well studied. Pathologic markers and staging systems have not been a precise predictor of the prognosis of an individual patient. Hence, we hypothesize to develop a transcript-based signature to categorize stage IIIA-NSCLC in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), plus identify markers that could indicate the prognosis of the disease. METHODS: Human Transcriptome Array 2.0 (HTA) and NanoString nCounter® platform were used for high-throughput gene-expression profiling. Initially, we profiled stage IIIA-NSCLC through HTA and validated through NanoString. Additionally, two metastatic markers SPP1 and CDH2 were validated in 47 NSCLC stage IIIA samples through real-time PCR. RESULTS: We observed distinct gene clusters in LUAD and LUSC with down-regulation of six genes and up-regulation of 57 genes through HTA. Ninety-six transcripts were randomly selected after analyzing HTA data and validated on the NanoString platform. We found 40 differentially expressed transcripts that categorized NSCLC into LUAD and LUSC. SPP1 is significantly overexpressed (4.311±1.27 fold in LUAD and 13.41±3.82 fold in LUSC compared to control), and the CDH2 transcript was significantly overexpressed (11.53 ± 4.027-fold compared to control) only in LUSC. DISCUSSION: These markers enable us to categorize stage IIIA NSCLC into LUAD and LUSC plus these markers may be helpful to understand the pathophysiology of NSCLC. However, more data required to make these findings useful in general clinical practice.

Differentially expressed full-length, fusion and novel isoforms transcripts-based signature of well-differentiated keratinized oral squamous cell carcinoma.

Highly keratinized oral squamous cell carcinoma (OSCC) exhibits an improved response to treatment and prognosis compared with weakly keratinized OSCC. Therefore, we aimed to develop gene transcript signature and to identify novel full-length isoforms, fusion transcript and non-coding RNA to differentiate well-differentiated (WD) with Moderately Differentiated (MD)/Poorly Differentiated (PD)/WD-lymphadenopathy OSCC through, HTA, Isoform sequencing, and NanoString. Additionally, specific copy number gain and loss were also identify in WD keratinized OSCC through Oncoscan array and validated through Real-time PCR in histopathologically characterized FFPE-WD keratinized OSCC. Three-hundred-thirty-eight (338) differentially expressed full-length (FL) transcript isoforms (317 upregulated and 21 down-regulated in OSCC) were identified through Isoform Sequencing using the PacBio platform. Thirty-four (34) highly upregulated differentially expressed transcripts from IsoSeq data were also correlated with HTA2.0 and validated in 42 OSCC samples. We were able to identify 18 differentially expressed transcripts, 12 fusion transcripts, and two long noncoding RNAs. These transcripts were involved in increased cell proliferation, dysregulated metabolic reprogramming, oxidative stress, and immune system markers with enhanced immune rearrangements, suggesting a cancerous nature. However, an increase in proteasomal activity and hemidesmosome proteins suggested an improved prognosis and tumor cell stability in keratinized OSCC and helped to characterize WD with MD/PD/WD with lymphadenopathy OSCC. Additionally, novel isoforms of IL37, NAA10, UCHL3, SPAG7, and RAB24 were identified while in silico functionally validated SPAG7 represented the premalignant phenotype of keratinized (K4) OSCC. Most importantly we found copy number gain and overexpression of EGFR suggest that TKIs may also be used as therapeutics in WD-OSCCs.

Draft genome assembly of Tenualosa ilisha, Hilsa shad, provides resource for osmoregulation studies.

This study provides the first high-quality draft genome assembly (762.5 Mb) of Tenualosa ilisha that is highly contiguous and nearly complete. We observed a total of 2,864 contigs, with 96.4% completeness with N50 of 2.65 Mbp and the largest contig length of 17.4 Mbp, along with a complete mitochondrial genome of 16,745 bases. A total number of 33,042 protein coding genes were predicted, among these, 512 genes were classified under 61 Gene Ontology (GO) terms, associated with various homeostasis processes. Highest number of genes belongs to cellular calcium ion homeostasis, followed by tissue homeostasis. A total of 97 genes were identified, with 16 GO terms related to water homeostasis. Claudins, Aquaporins, Connexins/Gap junctions, Adenylate cyclase, Solute carriers and Voltage gated potassium channel genes were observed to be higher in number in T. ilisha, as compared to that in other teleost species. Seven novel gene variants, in addition to claudin gene (CLDZ), were found in T. ilisha. The present study also identified two putative novel genes, NKAIN3 and L4AM1, for the first time in fish, for which further studies are required for pinpointing their functions in fish. In addition, 1.6 million simple sequence repeats were mined from draft genome assembly. The study provides a valuable genomic resource for the anadromous Hilsa. It will form a basis for future studies, pertaining to its adaptation mechanisms to different salinity levels during migration, which in turn would facilitate in its domestication.

Dysbiosis and Variation in Predicted Functions of the Granulation Tissue Microbiome in HPV Positive and Negative Severe Chronic Periodontitis.

Retrospective analysis has already shown correlation between severe Chronic Periodontitis (CP) cases with human papiloma virus (HPV). Hence, we aimed to explore deep-seated infected granulation tissue removed during periodontal flap surgery procedures for residential bacterial species between HPV+ and HVP- CP cases, which may serve as good predisposition marker for oral cancer. All CP-granulation samples showed the prominence of Firmicutes, Proteobacteria, and Bacteroidetes phyla with an abundance of gram negative anaerobes, except Streptococcus. In Beta diversity nonmetric multidimensional scaling plot, the random distribution of species was observed between HPV+ and HPV- CP granulation-samples. However, an abundance of Capnocytophaga ochracea was observed in HPV+ CP samples (p<0.05), while Porphyromonas endodontalis, Macellibacteroides fermentas, Treponema phagedenis, and Campylobacter rectus species were highly abundant in HPV- CP samples (p<0.05). The differential species richness leads altered functions related to mismatch-repair and nucleotide excision-repair and cytoskeleton-proteins. Hence, differential abundance of gram negative bacterial species between HPV+ and HPV- granulation-samples under anaerobic conditions may release virulence factors which may alter pathways favouring carcinogenesis. Hence, these species may serve as good predisposition marker for oral-cancer.

16S rRNA Long-Read Sequencing of the Granulation Tissue from Nonsmokers and Smokers-Severe Chronic Periodontitis Patients.

Smoking has been associated with increased risk of periodontitis. The aim of the present study was to compare the periodontal disease severity among smokers and nonsmokers which may help in better understanding of predisposition to this chronic inflammation mediated diseases. We selected deep-seated infected granulation tissue removed during periodontal flap surgery procedures for identification and differential abundance of residential bacterial species among smokers and nonsmokers through long-read sequencing technology targeting full-length 16S rRNA gene. A total of 8 phyla were identified among which Firmicutes and Bacteroidetes were most dominating. Differential abundance analysis of OTUs through PICRUST showed significant (p>0.05) abundance of Phyla-Fusobacteria (Streptobacillus moniliformis); Phyla-Firmicutes (Streptococcus equi), and Phyla Proteobacteria (Enhydrobacter aerosaccus) in nonsmokers compared to smokers. The differential abundance of oral metagenomes in smokers showed significant enrichment of host genes modulating pathways involving primary immunodeficiency, citrate cycle, streptomycin biosynthesis, vitamin B6 metabolism, butanoate metabolism, glycine, serine, and threonine metabolism pathways. While thiamine metabolism, amino acid metabolism, homologous recombination, epithelial cell signaling, aminoacyl-tRNA biosynthesis, phosphonate/phosphinate metabolism, polycyclic aromatic hydrocarbon degradation, synthesis and degradation of ketone bodies, translation factors, Ascorbate and aldarate metabolism, and DNA replication pathways were significantly enriched in nonsmokers, modulation of these pathways in oral cavities due to differential enrichment of metagenomes in smokers may lead to an increased susceptibility to infections and/or higher formation of DNA adducts, which may increase the risk of carcinogenesis.

Complete mitogenome of Indian mottled eel, Anguilla bengalensis bengalensis (Gray, 1831) through PacBio RSII sequencing.

Complete mitogenome sequence for Anguilla bengalensis bengalensis (family Anguillidae) was generated through third-generation sequencing platform. The 16 714 bp mitgenome sequence contained 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and a non-coding (control) region. The gene order was identical to that observed in most of the other vertebrates. The comparison of complete mitogenome sequence of Indian mottled eel generated during this study with two other subspecies did not agree with the taxonomic status of the three subspecies and considered as one species.

Expression profile and in silico characterization of novel RTF2h gene under oxidative stress in Indian catfish, Clarias magur (Hamilton 1822).

During the investigation of genes involved in the hypoxia tolerance, novel transcript, Replication Termination Factor 2 homologue (RTF2h), was found to be differentially expressed in brain of Clarias magur (previous name, C. batrachus) whose function was still undefined. Thus, present study was aimed to examine the transcriptional response of novel RTF2h gene, for its possible involvement in hypoxia tolerance in C. magur. Novel transcripts expressed under hypoxic stress were identified from ESTs obtained from SSH libraries of C. magur. Homology analysis of novel transcript (JK487668) revealed it to have RING superfamily signature and was closely related with RTF2 homologue. To characterize the expression pattern of CmRTF2h gene in different tissues of C. magur, qRT-PCR analysis was performed which showed significant increased expression, in spleen following short-term hypoxia exposure (12 H; 2.33-fold), while after long-term hypoxia exposure, significant up-regulation was found three tissues: in spleen (6.57-fold), liver (2.31-fold) and head kidney (2.99-fold) and was down-regulated in brain (3.52-fold). Further, the consensus sequence, obtained from SSH EST sequence and transcripts from hypoxia induced transcriptome data, through multiple sequence alignments, homology modeling and phylogentic analysis together, confirmed it as a novel CmRTF2h gene. In-silico protein-protein interaction and docking studies suggested its closely related function to that of RTF2. The present study reports the expression pattern of CmRTF2h under hypoxia. The up-regulated expression of CmRTF2h under hypoxic conditions may contribute to defense mechanism against induced DNA damage, which in turn may of adaptive significance for hypoxia tolerance in C. magur.

Hypoxic stress -responsive genes in air breathing catfish, Clarias magur (Hamilton 1822) and their possible physiological adaptive function.

The Indian catfish, Clarias magur (previous name C. batrachus) is an air breathing fish, inhabitant of aquatic bodies characterized by low dissolved oxygen levels. It is exposed to hypoxic conditions in its natural habitat. Thus, it can be useful model to study the mechanism of hypoxia stress tolerance. In C. magur, molecular processes facilitating its adaptation to hypoxia stress remain largely unexplored, in part due to unavailability of genomic resources. The suppression subtractive hybridization technique (SSH) was employed to compare the differential expression of transcripts under experimental hypoxic conditions, to that of normoxic conditions. Twelve subtracted cDNA libraries (six each forward and reverse) were constructed from brain, heart, liver, muscle, spleen and head kidney tissues. A total of 2020 clones were screened and sequenced, resulting into 1805 high quality expressed sequence tags (ESTs). Annotation of these differentially expressed ESTs resulted into the identification of genes involved in vast majority of pathways/processes affecting metabolism, cellular processes, signal transduction and/or immune functions. Additionally, 18 potential novel genes expressed in hypoxia stress exposed fish were also identified. The study had catalogued the differentially expressed genes from hypoxia stress induced C. magur, where most of them are reported for the first time in a hypoxia-tolerant fish species. The results not only provided insights for the hypoxia stress altered cellular functions in C. magur, but also generated a valuable functional genomics resource to assist targeted studies on functional genomics and future genome projects.

Hypoxia induced altered expression of heat shock protein genes (Hsc71, Hsp90α and Hsp10) in Indian Catfish, Clarias batrachus (Linnaeus, 1758) under oxidative stress.

Heat shock proteins (Hsps) are typically associated with stress response and tolerance. The Indian catfish, Clarias batrachus, is a freshwater air-breathing hypoxia tolerant teleost and is potentially important catfish species for aquaculture and for its economic value as food. The present study aimed at determining the transcriptional response of three Hsps, CbHsc71, CbHsp90α and CbHsp10 in hypoxia tolerant Indian catfish, C. batrachus, under experimental and natural hypoxia. The expression profile of above three genes were studied under different periods of hypoxia, through qRT-PCR. Primers were designed from ESTs obtained through SSH libraries constructed from hypoxia treated fishes. The Hsp10 ESTs and deduced protein was in silico characterized for its ORF and for its physical and chemical properties, respectively, using GeneScan, blastp, scanprosite, superfamily and other softwares. A phylogenetic tree was constructed based on deduced amino acid sequences of Hsc71, Hsp90α, Hsp90ß of Homo sapiens and other fishes along with CbHsp10 protein in MEGA4. The deduced protein sequences of CbHsp10 was found to have characteristic Hsp10 family signatures, and it is proposed for inclusion of methionine in the consensus sequences of Hsp10 family signature, after the "proline" residue. At transcription level, these genes were found to be differentially regulated under hypoxia stress, in different tissues of C. batrachus. The CbHsc71 and CbHsp90α were up-regulated after short and long-term hypoxia, whereas CbHsp10 was significantly down-regulated after short-term hypoxia. The differential expression of these Hsps may play a role in protection and survival under hypoxia induced oxidative stress in C. batrachus.

Hypoxia influences expression profile of Pleckstrin homology-like domain, family A, member 2 in Indian catfish, Clarias batrachus (Linnaeus, 1758): a new candidate gene for hypoxia tolerance in fish.

Several physiologically important genes were found to be regulated by hypoxia at the transcriptional level. The Pleckstrin homology-like domain, family A, member 2 (PHLDA2) gene was previously identified as an imprinted gene. The present study was aimed to determine the structure of complete cDNA and the deduced protein of PHLDA2 along with analysing the changes in its mRNA expression in Clarias batrachus tissues under hypoxic conditions. The complete cDNA of CbPHLDA2 gene consisted of 1009 nucleotides with an open reading frame of 417 nucleotides. The deduced CbPHLDA2 protein of 139 amino acids shared high homology with PHLD2A of other fishes as well as that of vertebrates. Importantly, a single amino acid (asparagine/lysine) insertion was identified in the PH domain of CbPHLDA2 and other fishes, which was absent in other vertebrates studied. Furthermore, under normoxic conditions, CbPHLDA2 was constitutively expressed with varying levels in analysed tissues. Short- and long-term hypoxia exposure resulted in significant changes in the expression of CbPHLDA2 in liver, spleen, head kidney, brain and muscle in a time-dependent manner. The results suggested that CbPHLDA2 might play an important role for adaptive significance under hypoxia.

Molecular characterization and expression analysis of three hypoxia-inducible factor alpha subunits, HIF-1α, -2α and -3α in hypoxia-tolerant Indian catfish, Clarias batrachus [Linnaeus, 1758].

The present study aimed at characterization of three HIF-α subunits, HIF-1α -2α and -3α from hypoxia-tolerant Clarias batrachus, as well as to elucidate their expression pattern under short and long-term hypoxic conditions and identification of biomarker candidate. The complete cDNAs of HIF-1α, -2α and -3α were 2,833, 4,270 and 3,256 bp in length, encoding 774, 818 and 628 amino acid residues, respectively. In C. batrachus, HIF-α subunits were structurally similar in DNA binding, dimerization, degradation and transcriptional activation domains, but differed in their oxygen-dependent degradation domains. Presence of c-Jun N-terminal kinase binding domain in HIF-α subunits was reported here for the first time in fish. In adult C. batrachus, three HIF-α mRNAs were detected in different tissues under normoxic conditions, however HIF-1α was highly expressed in all the tissues studied, in comparison to HIF-2α and -3α. Short-term hypoxia exposure caused significant increase in three HIF-α transcripts in brain, liver and head kidney, while after long-term hypoxia exposure, significant up-regulation of HIF-1α in spleen and -2α in muscle was observed and HIF-3α significantly down-regulated in head kidney. These observations suggest that the differential expression of HIF-α subunits in C. batrachus was hypoxic time period dependent and may play specialized roles in adaptive response to hypoxia. HIF-2α, with its highly elevated expression in muscle tissues, can be a robust biomarker candidate for exposure to hypoxic environment.

Molecular characterization and expression analysis of PPP1R3C in hypoxia-tolerant Indian catfish, Clarias batrachus (Linnaeus, 1758) under hypoxia.

Hypoxia is an important environmental stressor that leads to rapid adaptive changes in metabolic organization. However, the molecular mechanisms of hypoxia tolerance in fish remain largely unknown. The present work was focused on understanding the molecular mechanisms and signaling pathways that may lead to tolerance of Clarias batrachus to hypoxic stress. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) is a new hypoxia-inducible factor (HIF) targeted gene and is regulated by HIF-1 under hypoxic conditions. Overexpression of PPP1R3C increases glycogen accumulation through activation of several enzymes and processes. In this study, for the first time, full length cDNA of PPP1R3C from C. batrachus was characterized and its expression pattern in the brain, liver, muscle and spleen under short (progressive hypoxia; PH, 1h, 6h and 12h) and long-term (natural) hypoxic conditions was investigated. The complete cDNA of PPP1R3C was of 1499 bp, encoding 285 amino acid residues. The identified protein had a protein phosphatase 1 binding motif and a carbohydrate binding domain, thought to be involved in the regulation of glycogen metabolism. Short-term hypoxia exposure caused significant increase in PPP1R3C transcripts in the liver (6h; 6.96 fold and 12h; 3.91 fold) and muscle (progressive hypoxia; 3.46 fold), while, after long-term hypoxia exposure, significant up-regulation in the liver (7.77 fold) and spleen (6.59 fold) tissues was observed. No significant differences were observed in the brain for any time periods. Thus PPP1R3C may play an important role in the tolerance of C. batrachus to hypoxia.

Physiological responses to acute experimental hypoxia in the air-breathing Indian catfish, Clarias batrachus (Linnaeus, 1758).

With an aim to study the mechanism of adaptation to acute hypoxic periods by hypoxia-tolerant catfish, Clarias batrachus, the mass-specific metabolic rate (VO2) along with its hematological parameters, metabolic response and antioxidant enzyme activities were studied. During progressive hypoxia, C. batrachus was found to be an oxyconformer and showed a steady decline in its aquatic oxygen consumption rate. When C. batrachus was exposed for different periods at experimental hypoxia level (0.98 +/- 0.1 mg/L, DO), hemoglobin and hematocrit concentrations were increased, along with decrease in mean cellular hemoglobin concentration, which reflected a physiological adaptation to enhance oxygen transport capacity. Significant increase in serum glucose and lactate concentration as well as lactate dehydrogenase activity was observed. Antioxidant enzymes were found to operate independently of one another, while total glutathione concentration was unaffected in any of the tissues across treatments. These observations suggested that hypoxia resulted in the development of oxidative stress and C. batrachus was able to respond through increase in the oxygen carrying capacity, metabolic depression and efficient antioxidant defense system to survive periods of acute hypoxia.

Molecular characterization and expression analysis of a novel cystatin-like gene in a hypoxia-tolerant Indian catfish, Clarias batrachus [Linnaeus, 1758].

A novel member of Cystatin superfamily was identified from Indian catfish, Clarias batrachus, in response to oxidation stress induced by environmental hypoxia. Integrated genomic approaches, expression profiling and computational techniques showed that CbCystatin had putative cystatin/monelin like domain and might be a transmembrane and/or intermediate protein in signaling pathways. CbCystatin was found to be clustered into family 2 Cystatins. At transcriptional level, its expression was significantly up-regulated in response to short as well as long periods (more than 20 fold) of hypoxia, suggesting its positive association with oxygen concentrations lower than physiological concentrations.

Permanent genetic resources added to Molecular Ecology Resources Database 1 June 2012-31 July 2012.

This article documents the addition of 96 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Clarias batrachus, Marmota himalayana, Schizothorax richardsonii, Sitophilus zeamais and Syagrus romanzoffiana. These loci were cross-tested on the following species: Clarias dussumeri, Clarias gariepinus, Heteropneustus fossilis, Sitophilus granarius and Sitophilus oryzae.

Development of EST derived SSRs and SNPs as a genomic resource in Indian catfish, Clarias batrachus.

Clarias batrachus, an Indian catfish species, is endemic to the Indian subcontinent and potential cultivable species. The genomic resources in C. batrachus in the form of ESTs containing microsatellite repeats (EST-SSR) and single nucleotide polymorphisms (SNPs) that are associated with the expressed genes from spleen were mined. From a total of 1,937 ESTs generated, 1,698 unique sequences were obtained, out of which 221 EST-SSRs were identified and 54% could be functionally annotated by similarity searches. A total of 23 contigs containing 3 or more ESTs were found to contain 31 SNP loci, out of which 8 ESTs showed similarity to genes of known function and 1 for hypothetical protein. Nine ESTs with SSRs and/or SNPs identified in this study were reported to be associated with diseases in human and animals. These identified loci can be developed into markers in C. batrachus, which can be useful in linkage mapping, comparative genomics studies and for its genetic improvement programmes.