Variable Immunogenic Potential of Wheat: Prospective for Selection of Innocuous Varieties for Celiac Disease Patients via in vitro Approach.

Celiac Disease (CD) is a multifactorial, autoimmune enteropathy activated by cereal proteins in genetically predisposed individuals carrying HLA DQ2/8 genes. A heterogenous gene combination of the cereal prolamins is documented in different wheat genotypes, which is suggestive of their variable immunogenic potential. In the current study, four wheat varieties (C591, C273, 9D, and K78) identified via in silico analysis were analyzed for immunogenicity by measuring T-cell proliferation rate and levels of inflammatory cytokines (Interferon-γ and Tumor Necrosis Factor-α). Peripheral Blood Mononuclear Cells and biopsy derived T-cell lines isolated from four CD patients in complete remission and two controls were stimulated and cultured in the presence of tissue transglutaminase activated pepsin-trypsin (PT) digest of total gliadin extract from test varieties. The immunogenicity was compared with PBW 621, one of the widely cultivated wheat varieties. Phytohaemagglutinin-p was taken as positive control, along with unstimulated cells as negative control. Rate of cell proliferation (0.318, 0.482; 0.369, 0.337), concentration of IFN- γ (107.4, 99.2; 117.9, 99.7 pg/ml), and TNF- α (453.8, 514.2; 463.8, 514.2 pg/ml) was minimum in cultures supplemented with wheat antigen from C273, when compared with other test varieties and unstimulated cells. Significant difference in toxicity levels among different wheat genotypes to stimulate celiac mucosal T-cells and PBMC's was observed; where C273 manifested least immunogenic response amongst the test varieties analyzed.

Deciphering the transcriptome of prepubertal buffalo mammary glands using RNA sequencing.

Although water buffaloes are the main milk-producing animals in Indian subcontinent, only limited attempts have been made to identify canonical pathways and gene regulatory networks operating within the mammary glands of these animals. Such information is important for identifying unique transcriptome signatures in the mammary glands of diseased animals. In this report, we analyzed the transcription profile of 3 prepubertal buffalo mammary glands and identified common genes (mean FPKM > 0.2 in all samples) operating in the glands. Among 19,994 protein coding genes, 14,678 genes expressed and 5316 unique genes did not express in prepubertal buffalo mammary glands. Of these 14,678 expressed genes, 79% comprised a ubiquitous transcriptome that was dominated by very lowly expressed genes (51%). The percentage of rarely, moderately, and abundantly expressed genes was 25%, 2%, and 1%, respectively. Gene Ontology (GO) terms reflected in the expression of common genes (mean FPKM > 5.0) for molecular function were related to binding and catalytic activity. Products of these genes were involved in metabolic and cellular processes and belong to nucleic acid binding proteins. The canonical pathways for growth of mammary glands included integrin signaling, inflammation, GnRH and Wnt pathways. KEGG enriched pathways revealed many pathways of cancer including ribosome, splisosome, endocytosis, and ubiquitin-mediated proteolysis, pathways for viral infection, and bacterial invasion of epithelial. Highly expressed genes (mean FPKM > 500 included beta-actin (ACTB), beta-2 microglobulin (B2M), caseins (CSN2, CNS3), collagens (COL1A1, COL3A1), translation elongation factors (EEF1A1, EEF1G, EEF2), keratins (KRT15, KRT19), major histocompatibility complex genes (CD74, JSP.1), vimentin (VIM), and osteopontin (SPP1). Interestingly, expression of milk protein genes in prepubertal glands opens possible roles of these genes in development of mammary glands. We report the whole transcriptomic signature of prepubertal buffalo mammary gland and indicated its molecular signature is similar to cancer type.

Long-term exposures to ethion and endotoxin cause lung inflammation and induce genotoxicity in mice.

Ethion, an organophosphorus pesticide, is used worldwide and has potential for toxicity and inflammation. There are very limited data on the pulmonary and genotoxic effects of ethion especially when the exposure is combined with lipopolysaccharide. Therefore, we used a mouse model to test the hypothesis that prolonged exposure to ethion alone or in conjunction with lipopolysaccharide (LPS) will cause lung inflammation and genotoxicity in a mouse model. Swiss albino (n = 30) were divided into a control (n = 10) and two treatment groups (n = 10; each group). The treatment groups were orally administered ethion (4 or 2 mg/kg/animal/day; n = 10 each) dissolved in corn oil for 90 days. After 90 days of exposure, five animals from each of the groups were challenged with 80 µg Escherichia coli lipopolysaccharide (LPS) intranasally and the remaining five animals with normal saline solution via the same route. Ethion at both dosages induced lung inflammation as indicated by increased (p < 0.05) perivascular and peribronchial accumulation of inflammatory cells along with thickening of the alveolar septal wall. Ethion at 4 mg/kg altered (p < 0.05) the mRNA and protein expression of TLR-9 and IL-1ß in the lungs and induced genotoxicity in blood cells as determined by single cell gel electrophoresis (Comet assay). Further, both dosages of ethion in combination with E. coli LPS caused genotoxicity and increased (p < 0.05) pulmonary expression of TLR-4, TLR-9 and IL-1ß. The data taken together suggest ethion induces lung inflammation and interaction between ethion and LPS increases genotoxicity in blood cells.

Long non-coding RNA: its evolutionary relics and biological implications in mammals: a review.

The central dogma of gene expression propounds that DNA is transcribed to mRNA and finally gets translated into protein. Only 2-3% of the genomic DNA is transcribed to protein-coding mRNA. Interestingly, only a further minuscule part of genomic DNA encodes for long non-coding RNAs (lncRNAs) which are characteristically more than 200 nucleotides long and can be transcribed from both protein-coding (e.g. H19 and TUG1) as well as non-coding DNA by RNA polymerase II. The lncRNAs do not have open reading frames (with some exceptions), 3`-untranslated regions (3'-UTRs) and necessarily these RNAs lack any translation-termination regions, however, these can be spliced, capped and polyadenylated as mRNA molecules. The flexibility of lncRNAs confers them specific 3D-conformations that eventually enable the lncRNAs to interact with proteins, DNA or other RNA molecules via base pairing or by forming networks. The lncRNAs play a major role in gene regulation, cell differentiation, cancer cell invasion and metastasis and chromatin remodeling. Deregulation of lncRNA is also responsible for numerous diseases in mammals. Various studies have revealed their significance as biomarkers for prognosis and diagnosis of cancer. The aim of this review is to overview the salient features, evolution, biogenesis and biological importance of these molecules in the mammalian system.

Over-expression of mammaglobin-B in canine mammary tumors.

BACKGROUND: Mammaglobin, a member of secretoglobin family has been recognized as a breast cancer associated protein. Though the exact function of the protein is not fully known, its expression has been reported to be upregulated in human breast cancer.We focused on studying the expression of mammaglobin-B gene and protein in canine mammary tumor (CMT) tissue. Expression of mammaglobin-B mRNA and protein were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), respectively. RESULTS: High levels of mammaglobin-B mRNA expression (6.663 ± 0.841times) was observed in CMT as compared to age and breed matched healthy controls. Further, expression of mammaglobin-B protein was detected in paraffin-embedded mammary tumor tissues from the same subjects by IHC. Mammaglobin-B protein was overexpressed only in 6.67% of healthy mammary glands while, a high level of its expression was scored in 76.7% of the CMT subjects. Moreover, no significant differences in terms of IHC score and qRT-PCR score with respect to CMT histotypes or tumor grades were observed, indicating that mammaglobin-B over-expression occurred irrespective of CMT types or grades. CONCLUSION: Overall, significantly increased expression of mammaglobin-B protein was found in CMTs with respect to healthy mammary glands, which positively correlates to its transcript. These findings suggest that overexpression of mammaglobin-B is associated with tumors of canine mammary glands.

In vivo response of xanthosine on mammary gene expression of lactating Beetal goat.

Xanthosine is hypothesized to increase stem cell number by promoting symmetrical cell division. Stem cells, in particular mammary stem/progenitor cells are important for gland growth and tissue repair. Molecular mechanism of xanthosine effects on mammary tissue is very limited therefore, a detailed study is warranted. The objective of this study was to evaluate transcriptomic changes in mammary gland infused/not infused with xanthosine of lactating goat. Seven primiparous Beetal goats on day 5 after kidding, were selected for the study. One gland of each goat was infused with xanthosine (TRT gland) twice daily for 3 days while the other gland did not receive any xanthosine and served as control (CON gland). Biopsy of mammary tissues was taken from TRT and CON glands, 2 days after the last day of treatment that is on day 10 after kidding. Illumina RNA-sequencing (RNA-seq) was performed for global gene expression analysis of contralateral glands. Of 382 differentially expressed genes (DEGs), 372 genes were annotated to the goat genome. Gene ontology analyses revealed majority of the DEGs to be associated with metabolic pathways (glycan and lipid metabolism), biosynthesis of antibiotics and peroxisome proliferator-activated receptor signalling pathways. These molecular pathways are either directly or indirectly involved with lipid metabolism in mammary tissue and host adaptive immune response. Expression of stem cell marker namely aldehyde dehydrogenase enzymes (ALDH1A1, ALDH3B1) were upregulated in the treatment gland. Real-time quantitative PCR (RT-qPCR) analyses of selected DEGs showed their expression profiles to be in agreement with results of RNA-seq. To our knowledge, this is the first study that describes effects of xanthosine on transcriptomic changes of mammary tissue. This information can be used further to dissect the molecular mechanisms underlying effects of xanthosine to improve production potential and udder health.

The overexpression of Hsp90B1 is associated with tumorigenesis of canine mammary glands.

Heat shock proteins (Hsp) are molecular chaperones that are responsible for protein folding and maintenance of cellular homeostasis. Hsp90, an important member of HSP family, has an important role in breast cancer. Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog of Hsp90 encoded by Hsp90B1 gene. To test if this protein is overexpressed in dogs with mammary tumor, we estimated and compared its serum levels in healthy dogs and that of dogs with mammary tumors. Hsp90B1 mRNA expression was measured in tumorous and healthy mammary tissues (from age- and breed-matched dogs) by real-time PCR. The gene was found to be overexpressed in mammary tumors (3.586 ± 0.067 times). Further, it was heterologously expressed in a prokaryotic system as 90 kDa protein. A recombinant Grp94-based sandwich ELISA was developed to quantify serum Grp94 in dogs with mammary tumors. Based on receiver-operating characteristics' analysis, the assay was found to be 90.62% sensitive and 93.75% specific for a cutoff value of 0.35 with respect to histopathological staining in diagnosing the disease. The t test showed that serum Grp94 levels were significantly elevated (92.97 ± 3.62 ng/ml) in dogs with mammary tumors compared with healthy controls (10.30 ± 0.79 ng/ml) (p < 0.0001). These findings suggest that Grp94 might act as a potential biomarker for prognosis of canine mammary tumors and monitoring its therapy.

Anticancer potential of dietary vitamin D and ascorbic acid: A review.

Cancers have been the leading cause of death worldwide and poor diet and physical inactivity are major risk factors in cancer-related deaths. Micronutrients such as vitamins and minerals appear to have preventive properties against cancer. One important mechanism by which dietary changes can exert preventive effects on cancer is via the modulation of micronutrient concentrations in target tissues. Many of these micronutrients are available in the form of dietary supplements, and the intake of these supplements is prevalent in various parts of the world. However, in most cases, it is not known which micronutrient (or combination of micronutrients) is best when it comes to lowering the risk of cancer. The present review illustrates the effect of vitamin D and ascorbic acid intake on preventing cancer.

Development of recombinant matrix metalloproteinase-3 based sandwich ELISA for sero-diagnosis of canine mammary carcinomas.

Matrix metalloproteinase-3 is invariably upregulated in cancerous condition. So we aimed to determine serum level of MMP-3 in canine mammary tumors. The gene was expressed in E. coli system as ~43kDa recombinant protein, which was refolded, purified, and confirmed. Hyperimmune serum was raised against the expressed protein in rabbits and mice to standardize sandwich ELISA. ROC analysis revealed largest area under the curve of 0.998 with sensitivity (100%) and specificity (95%) for a cut-off value of 0.363 with respect to histopathological staining. The finding of the present study indicates that MMP-3 can act as a potential molecular marker for serodiagnosis of canine mammary carcinomas.

Mammaglobin as a diagnostic serum marker of complex canine mammary carcinomas.

Mammaglobin is a glycoprotein exhibiting homology to uteroglobin gene family. Although the biological function of the protein is not yet known it has been reported to act as marker for breast cancer in women. This study reports the expression of mammaglobin gene in canine mammary tumor condition. The gene was cloned, sequenced and heterologously expressed in Escherichia coli host system as 12 kDa (approx.) recombinant fusion protein. The expressed protein was further purified to homogeneity and confirmed by western blotting. Hyperimmune sera were raised against the expressed protein in rabbits and mice to standardize sandwich ELISA for relative quantification of circulating protein in the sera of dogs with mammary tumors. Based on receiver-operating characteristics analysis, the test was found to be 90% sensitive and 95% specific for a cut-off value of 0.177 with respect to histopathological staining in diagnosing canine mammary tumors and the protein level was not elevated in other diseased conditions. These findings indicate that it can act as a novel molecular marker for detecting mammary tumors in canines.