Delay in puberty is dependent on heat shock protein B1 expression in native cross layers of Punjab under heat stress.

Small heat shock protein B1 (HspB1) has been reported to play an essential role in thermotolerance. This study aimed to determine a correlation (if any) between HspB1 expression and age at first egg lay (puberty) in a native cross-layer poultry of Punjab under heat stress. Forty native cross-layer birds were reared in two different seasons, viz. summer (THI was more than 27), classified as the heat-stressed group (n = 20) and winter season (THI was less than 21), classified as the control group (n = 20). Blood was collected from both the groups of birds in their 15th week of age and at puberty. Serum catalase and superoxide dismutase activities, reduced glutathione and corticosterone concentration and lipid peroxidation were measured to assess the oxidative stress in both the groups of birds. The serum antioxidants significantly decreased whilst corticosterone levels and lipid peroxidation significantly elevated in birds in response to summer heat stress. Moreover, in summer season, the activities of the antioxidant enzymes further decreased and lipid peroxidation further increased significantly in birds from their pre-pubertal stage to puberty, which was not observed during the winter season. A clone of chicken HspB1 in BL21 (DE3) cells was revived, and recombinant HspB1 was purified using Ni-NTA agarose column. Serum HspB1 concentration was estimated in different groups of birds by indirect ELISA that has been standardized using the recombinant chicken HspB1. Compared to the control, birds under heat stress had significantly higher serum HspB1 levels. The delay in puberty of all the heat-stressed birds was significantly associated with the increase in their serum HspB1 levels. Taken together, the expression of HspB1 was found to be associated with age at puberty in the native cross poultry layers of Punjab.

Exposures to 2,4-Dichlorophenoxyacetic acid with or without endotoxin upregulate small cell lung cancer pathway.

BACKGROUND: Pesticide residues in food and environment along with airborne contaminants such as endotoxins pose health risk. Although herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) has been associated with increased risk of lung cancers such as small cell lung cancer (SCLC) among agricultural workers, there are no data on the SCLC signaling pathway upon 2,4-D exposure without LPS or in combination with endotoxin. METHODS: We exposed Swiss albino mice (N = 48) orally to high (9.58 mg kg- 1) and low (5.12 mg kg- 1) dosages of 2,4-D dissolved in corn oil for 90 days followed by E. coli lipopolysaccharide (LPS) or normal saline solution (80 µl/animal). Lung samples and broncho-alveolar fluid (BALF) were subjected to Total histological score (THS) and total leucocyte count (TLC) and differential leucocytes count (DLC) analyses, respectively. We used microarray and bioinformatics tools for transcriptomic analyses and differentially expressed genes were analyzed to predict the top canonical pathways followed by validation of selected genes by qRT-PCR and immunohistochemistry. RESULTS: Total histological score (THS) along with BALF analyses showed lung inflammation following long term dietary exposure to high or low doses of 2,4-D individually or in combination with LPS. Microarray analysis revealed exposure to high dose of 2,4-D without or with LPS upregulated 2178 and 2142 and downregulated 1965 and 1719 genes, respectively (p < 0.05; minimum cut off 1.5 log fold change). The low dose without or with LPS upregulated 2133 and 2054 and downregulated 1838 and 1625 genes, respectively. Bioinformatics analysis showed SCLC as topmost dysregulated pathway along with differential expression of Itgb1, NF-κB1, p53, Cdk6 and Apaf1. Immunohistological and quantitative real time PCR (qRT-PCR) analyses also supported the transcriptomic data. CONCLUSIONS: Taken together, the data show exposures to high and low dose of 2,4-D with/without LPS induced lung inflammation and altered pulmonary transcriptome profile with the involvement of the SCLC pathway. The data from the study provide the insights of the potential damage on lungs caused by 2,4-D and help to better understand the mechanism of this complex relation.

Expression of recombinant DnaK of Brucella abortus and its evaluation as immuno-modulator.

Heat shock proteins are molecular chaperones that are immunogens as well as potent inducers of an antigen-specific immunological response. In this study, we aimed to evaluate if co-immunization of Brucella rOmp22 and rDnaK proteins had boosted immunogenic activity as compared to rOmp22 immunization alone in mice. For this, gene-encoding DnaK of B. abortus was cloned, expressed in E. coli and purified using Ni-NTA agarose. Immuno-modulatory effect of rDnaK protein was evaluated in mice when co-immunized with Brucella rOmp22. Four groups of mice (n = 6 per group) were used in the study. The control group was immunized with rOmp22 alone, while rOmp22 emulsified with conventional adjuvants (Freund's complete and incomplete adjuvants) and rOmp22 mixed with rDnaK were injected to group I and group II in mice, respectively. Group III mice were immunized with rDnaK alone. IgG class switching (IgG1 and IgG2a) response to immunization was assessed by enzyme-linked immunosorbent assay and expression of IL-4 and IL-12 mRNA was assessed by real-time PCR to evaluate the immune response in mice. The ratio of IgG1-IgG2a was less than 1 in mice co-immunized with rOmp22 and rDnaK, indicating that the immune response was directed towards CMI arm in this group of mice. Moreover, IL-12 mRNA expression was also up-regulated to a greater extent in mice co-immunized with rOmp22 and rDnaK as compared to those immunized with rOmp22 along with the conventional adjuvants, or rOmp22 alone. Our data suggest that rDnaK could be responsible for modulating the immune response, specifically the CMI response.

Heat shock protein B1 expression is associated with age at sexual maturity in Rhode Island Red and Punjab Red layers under heat stress.

Heat shock proteins (Hsp) aid in protein folding and also to combat stress in all cellular organisms. HspB1 is a member of the small HSP family that has a significant role in thermo-tolerance. In this study, we aimed to determine the relationship (if any) between age at sexual maturity of layer poultry (Rhode Island Red and Punjab Red) and HspB1 expression both at mRNA and protein levels under heat stress. The mRNA expression of hspB1 was checked by real-time PCR. Delay in sexual maturity of the birds was found to be directly associated with the hspB1 mRNA expression in both the bird varieties under heat stress. No significant regression (association) of hspB1 mRNA expression with age at sexual maturity was observed in case of control, non-heat stressed birds. The serum levels of HspB1 were measured by indirect ELISA, using recombinant HspB1 that was expressed using pET-32b(+) vector in BL21(DE3) cells. Serum HspB1 concentration increased significantly (p ≤ 0.001) in heat-stressed birds as compared with control ones. A significant association was found between the increase in serum HspB1 concentration and delay in sexual maturity of all the birds under heat stress while no such association was found in control birds. In conclusion, HspB1 mRNA and protein expression were found to be associated with age at sexual maturity in Punjab Red and RIR layers under heat stress.

Circulating level of heat shock protein 27 is elevated in dogs with mammary tumors.

In the current study, we have synthesized canine recombinant Hsp27 in E. coli and raised hyperimmune serum against the protein in mice. Using the hyperimmune serum, an indirect ELISA was developed to estimate circulating levels of Hsp27 in dogs with various types of mammary neoplasia and to compare their levels with those of tumor-free apparently healthy dogs. The developed assay had a high specificity (83.3%) and sensitivity (96.6%) for a cut-off value of 0.45 with respect to histopathological examination in discriminating healthy dogs from those bearing mammary tumors. Serum Hsp27 levels were found to be significantly elevated in tumor subjects (20.33 ± 1.23 ng/ml) as compared to healthy controls (6.56 ± 0.51 ng/ml) and the increase in the Hsp27 levels was irrespective of mammary tumor histotypes. However, dogs with grade-II tumors had higher Hsp27 levels as compared to grade-I types. Therefore, Hsp27 can be exploited as one of the 'neoplastic signatures' of canine mammary tumors.

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.

Proteomic Characterization of Lytic Bacteriophages of Staphylococcus aureus Isolated from Sewage Affluent of India.

Staphylococcus aureus is a Gram-positive bacterium that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to need for an alternative treatment such as bacteriophage therapy. Present study describes isolation and characterization of a staphylococcal phage from sewage samples. S. aureus isolates obtained from microbial type culture collection (MTCC), Chandigarh, India, were used to screen staphylococcal phages. A phage designated as ΦMSP was isolated from sewage samples by soft agar overlay method. It produced clear plaques on tryptone soya agar overlaid with S. aureus. Transmission electron microscopy revealed that the phage had an icosahedral symmetry. It had 5 major proteins and possessed a peptidoglycan hydrolase corresponding to 70 kDa. ΦMSP infection induced 26 proteins to be uniquely expressed in S. aureus. This phage can be proposed as a candidate phage to treat staphylococcal infections.

Cyclical changes in collagen concentration in relation to growth and development of buffalo corpus luteum.

In the present study, changes in luteal fresh weight and concentration of collagen in cyclic buffalo corpus luteum were investigated at 4 stages of its growth and development/regression. The collagen concentration was determined by estimating hydroxyproline, a collagen specific amino acid present in luteal tissues. The mean fresh weight increased (P < 0.001) over the luteal phase, reached maximum at late-luteal stage and decreased (P < 0.001) subsequently at follicular stage. The weight of the mature CL was 2.5 times heavier than the CL haemorrhagicum and regressing CL. Results showed that cyclic buffalo CL contains collagen at all 4 stages of development during oestrous cycle. The collagen in luteal tissues constitutes about 0.9% to 1.2% of luteal fresh weight with the highest content appearing in mature tissue. The concentration of collagen increased (P < 0.001) with the stages of CL development over the luteal phase and the highest concentration was recorded at follicular phase with the decline of luteal weight. The total content of collagen per CL also showed the same trend during luteal phase but decreased at follicular phase with the loss of luteal tissues. In conclusion, collagen concentration in cyclic buffalo CL changes with the growth and development of CL across the oestrous cycle. The synthesis of collagen is faster between early- to mid-luteal stage than mid- to late-luteal stage.