We report detection of Baculoviral inhibitor of apoptosis repeat containing-5 (BIRC5) protein biomarker in dog serum by label-free surface plasmon resonance (SPR) immunosensor. Initially, overexpression of BIRC5 in canine mammary tumour (CMT) tissues was confirmed by real-time PCR. Recombinant BIRC5 was produced and protein specific antibodies developed in guinea pig specifically reacted with native protein in immunohistochemistry and immunocytochemistry. SPR immunosensor was developed by fabricating anti-BIRC5 antibodies on gold sensor disc. The equilibrium dissociation constant, (KD = kd/ka) was 12.1 × 10−12 M; which indicates that antibodies are of high affinity with sensitivity in picomolar range. The SPR assay could detect as low as 6.25 pg/ml of BIRC5 protein in a calibration experiment (r2 = 0.9964). On testing real clinical samples, 95% specificity and 73.33% sensitivity were recorded. The average amount of serum BIRC5 in dogs with CMT was 110.02 ± 9.77 pg/ml; whereas, in non-cancerous disease conditions, 44.79 ± 4.28 pg/ml and in healthy dog sera 30.28 ± 2.99 pg/ml protein was detected. The SPR immunosensor for detection of BIRC5 in dog sera is reported for the first time and this may find prognostic and diagnostic applications in management of CMT. In future, ‘on-site’ sensors can be developed using this technique for near-patient testing.

Associations between abnormal methylation of spermatozoan DNA with male infertility have been sought in recent years to identify a molecular explanation of differential spermatozoan function. The present work was undertaken to investigate the methylation profile of differentially methylated regions (DMRs) in the IGF2-H19 locus of Bos taurus X Bos indicus crossbred bull spermatozoa. Bulls having more than at least 100 insemination records over a period of 12 years were classified into two groups of five bulls each belonging to low- and high-fertility groups. The IGF2 and H19 DMR sequences in B. indicus cattle were observed to be in absolute homology with B. taurus cattle. The DNA of crossbred bull spermatozoa was isolated, bisulfite treated, and amplified for specific DMR regions using methylation-change-specific primers. The overall degree of methylation at IGF2-H19 DMRs was not found to be significantly different among two groups of bulls. The sixth CTCF binding site (CCCTC) identified in H19 DMR, however, had a significant methylation difference between the high- and low-fertility bulls. It was concluded that alteration of the methylation levels at IGF2-H19 DMRs might not be responsible for the fertility difference of crossbred bulls, although the role played by the specific CTCF binding sites at this locus, which could influence IGF2 expression during spermatogenesis and early embryonic development, deserves further attention.

Spontaneously occurring canine mammary tumours (CMTs) are the most common neoplasms of female unspayed dogs and are of potential importance as models for human breast cancer as well. Mortality rates are thrice higher in dogs as compared to humans with breast cancer, which can partly be attributed to lack of diagnostic techniques for their early detection. Human breast cancer studies reveal role of autoantibodies in early cancer diagnosis and also the usefulness of autoantibody panels in increasing the sensitivity, as well as, specificity of diagnostic assays. Therefore, in this study, we took advantage of high-throughput Luminex technique for developing a multiplex assay to detect autoantibody signatures against 5 canine mammary tumour-associated autoantigens (TAAs). These TAAs were expressed separately as fusion proteins with halo tag at the N-terminus, which allows easy and specific covalent coupling with magnetic microspheres. The multiplex assay, comprising a panel of candidate autoantigens (TPI, PGAM1, MNSOD, CMYC & MUC1) was used for screening circulating autoantibodies in 125 dog sera samples, including 75 mammary tumour sera and 50 healthy dog sera. The area under curve (AUC) of the combined panel of biomarkers is 0.931 (p < 0.0001), which validates the discriminative potential of the panel in differentiating tumour patients from healthy controls. The assay could be conducted in 3hrs using only 1ul of serum sample and could detect clinical cases of canine mammary tumour with sensitivity and specificity of 78.6% and 90%, respectively. In this study, we report for the first time a multiplexed assay for detection of autoantibodies in canine tumours, utilizing luminex technology and halo-tag coupling strategy. Further to the best of our knowledge, autoantibodies to CMYC and MUC1 have been reported for the first time in canines in this study.

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The BIRC 5 (also called survivin), member of the inhibitor of apoptosis protein family is highly over-expressed in human and animal cancers leading to poor prognosis. Still there is limited information about the gene sequence in dogs suffering with canine mammary tumour. Therefore, the present study was undertaken to find out any correlation of BIRC 5 gene over-expression with mutation status of the gene. The CMT tissues were confirmed by histopathological examination and included cases of mixed myoepithelioma, complex adenocarcinoma, mixed mammary capillary cystic adenocarcinoma and invasive solid carcinoma etc. Quantitative Real Time PCR (qRT-PCR) revealed 5.6±0.462-60.0±1.476 fold higher BIRC 5 gene expression levels in CMT tissues as compared to dog normal mammary gland tissues. The coding region of the gene was amplified, cloned and sequenced from a case of complex mammary carcinoma showing approximately 60.0±1.476 fold amplification of BIRC 5 gene. The sequence showed 100% similarity with the mRNA sequences of normal dog BIRC 5 present in NCBI. This indicates that BIRC 5 gene sequence in healthy dogs is similar to dogs suffering from CMT and showing over-expression of the gene. The multiple sequence alignment of the survivin gene with other species like cat, cow, buffalo, sheep, goat and human etc. revealed more than 90% similarity. The phylogenetic analysis demonstrates that gene is highly conserved across species to maintain its functional integrity. The findings revealed that there is no sequence alteration in BIRC 5 gene sequence in the CMT tissue showing more than 60 fold over-expression of the gene.

M atrix metalloproteinases (MMPs) encompasses a family of secreted, calcium dependent, zinc containing endopeptidases. These enzymes are responsible for tissue remodelling and are capable of degrading all kinds of extracellular matrix proteins including collagens, matrix glycoproteins, gelatin, elastins, and proteoglycans. MMPs are involved in regulation of cell behaviours like cellular differentiation, proliferation, angiogenesis, invasion, and apoptosis. Thus, these enzymes have multi-factorial roles during tumor progression: promoting establishment, tumor cell exfoliation, invasion and angiogenesis. The degradation of extracellular matrix components is crucial for tumor cell invasion and MMPs, especially MMP2 (72 kDa gelatinase A) and MMP9 (92 kDa gelatinase B), play critical role in degradation of gelatin and type IV collagen, the two major constituents of extracellular matrix. MMP9 and MMP2 are involved in invasion and metastases of many human cancers such as cancers of colon (Karakiulakis et al., 1997), breast (Bachmeier et al., 2001), brain tumours (Pagenstecher et al., 2001), prostate cancer (Brehmer et al., 2003), cervical cancer (Sato et al., 2004), melanoma (Bérubé et al., 2005) and many others. Further these ge-Short Communication Abstract | Matrix Metalloproteinases (MMP) 2 & 9 are overexpressed in wide variety of cancers and play important roles in tumour cell proliferation, invasion and metastasis. Tissue and serum levels of MMP2 and MMP9 correlate with disease prognosis. Real-time PCR is emerging as an alternative or supplementary technique to immunohisto-chemistry (IHC) for sensitive detection of tumour markers in tissues. Therefore, in this study real-time PCR assays were standardized for detecting over-expression of MMP2 and MMP9 in canine mammary tumour (CMT). Primers designed for real-time PCR analysis of MMP2 & MMP9 were specific as indicated by amplification plots and melt-curve analysis of amplicons. These amplicons showed sharply defined melting curves with single peaks at expected melting points. The over-expression of MMP2 and MMP9 was examined in 12 CMT tissues by real-time PCR analysis. Over-expression of MMP2 & MMP9 was observed in 58.3(7/12) and 41.6 % (5/12) cases of CMT respectively. Expression levels of MMP2 were 2.23±0.10 to 29.80±3.5 fold higher and for MMP2, 1.5±0.56 to 22.67±2.35 fold higher in cases of CMT than normal mammary gland biopsy. Approximately 33.3 % (4/12) of CMT tissues showed co-expression of both MMP2 and MMP9. Further studies are required to correlate MMP2 and MMP9 expression levels with CMT prognosis.

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