Urinary Cell-Free miR-99a-5p as a Potential Biomarker for Estrus Detection in Buffalo.

Accurate estrus detection method is the need of the hour to improve reproductive efficiency of buffaloes in dairy industry, as the currently available estrus detection methods/tools lack high sensitivity and specificity. Recently, circulating miRNAs have been shown as non-invasive biomarkers by various studies. Hence, in order to evaluate their potential as estrus biomarkers, the objective of this study was to identify and compare the levels of 10 hormone-responsive miRNAs in the urine collected at proestrus (PE), estrus (E), and diestrus (DE) phases of buffaloes (n = 3) pertaining to a discovery sample. Among 10 urinary miRNAs, the levels of bta-mir-99a-5p (E/PE 0.5-fold, P < 0.05; DE/PE 1.9-fold), bta-miR-125b (E/PE 0.5-fold; DE/PE 0.7-fold), bta-mir-145 (E/PE 1.5-fold; DE/PE 0.7-fold), bta-mir-210 (E/PE 1.2-fold, DE/PE 0.7-fold), mir-21 (E/PE 1.5-fold, DE/PE 2-fold), and bta-mir-191 (E/PE 1.3-fold; DE/PE 0.8-fold) were found to be altered during different phases of buffalo estrous cycle. In contrast, bta-mir-126-3p, bta-let-7f, bta-mir-16b, and bta-mir-378 were undetected in buffalo urine. Furthermore, a validation study in an independent group of 25 buffalo heifers showed the increased levels of urinary bta-mir-99a-5p during the DE (3.92-fold; P < 0.0001) phase as compared to the E phase. Receiver operating characteristic curve analyses also revealed the ability of urinary miR-99a-5p in distinguishing the E from the DE phase (area under the curve of 0.6464; P < 0.08). In silico analysis further showed an enrichment of miR-99a-5p putative targets in various ovarian signaling pathways, including androgen/estrogen/progesterone biosynthesis and apoptosis signaling, implicating the role of miR-99a-5p in ovarian physiology. In conclusion, significantly lower levels of bta-mir-99a-5p at the E phase than the DE phase in buffalo urine indicate its biomarker potential, which needs to be further explored in a large cohort in the future studies.

Validation of salivary ferning based estrus identification method in a large population of water buffaloes (Bubalus bubalis) using Foldscope.

Missing an estrus event is an economic problem in buffaloes because of lack of a simple and accurate estrus identification method. Saliva, a non-invasive fluid available every time, showed typical fern-like crystallization patterns at early estrus in buffaloes. However, to implement this salivary ferning based estrus identification method in the field conditions, the present study, for the first time, validated this method in four buffalo population samples (PS) representing four different field scenarios, an organized herd (PS1, 10 buffaloes monitored daily for a year (149 estrus events)), artificial insemination (AI) centers (PS2, 114 buffaloes brought for AI), induced estrus condition (PS3, 44 buffaloes) and farmers' doorsteps (PS4, 275 random buffaloes with unknown reproductive history and no estrous signs). Totally, 582 saliva samples were collected from 443 buffaloes. Salivary smears were observed under a simple microscope and/or a less expensive (< 1USD) paper microscope, Foldscope. On the basis of salivary fern-like patterns, the proportions of estrus identification were significantly different among PS. Specifically, the proportions in the PS1 (0.74, P < 0.0001) and PS4 (0.08, P < 0.05) were significantly higher than their population proportion estimates, 0.5 and zero, respectively. Therefore, this estrus identification method is much useful in the PS1 and PS4, the practical field scenarios requiring an accurate estrus prediction method, compared to the PS2 and PS3. Especially, this method is 91 % confirmatory to detect early estrus in PS4. Therefore, salivary ferning is a useful technique to identify early estrus in buffaloes in the field conditions at farmers' doorsteps.

Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.

Astrocytoma is the most common type of brain cancer constituting more than half of all brain tumors. With an aim to identify markers describing astrocytoma progression, we have carried out microarray analysis of astrocytoma samples of different grades using cDNA microarray containing 1152 cancer-specific genes. Data analysis identified several differentially regulated genes between normal brain tissue and astrocytoma as well as between grades II/III astrocytoma and glioblastoma multiforme (GBM; grade IV). We found several genes known to be involved in malignancy including Achaete-scute complex-like 1 (Drosophila) (ASCL1; Hash 1). As ASCL has been implicated in neuroendocrine, medullary thyroid and small-cell lung cancers, we chose to examine the role of ASCL1 in the astrocytoma development. Our data revealed that ASCL1 is overexpressed in progressive astrocytoma as evidenced by increased levels of ASCL1 transcripts in 85.71% (6/7) of grade II diffuse astrocytoma (DA), 90% (9/10) of grade III anaplastic astrocytoma (AA) and 87.5% (7/8) of secondary GBMs, while the majority of primary de novo GBMs expressed similar to or less than normal brain levels (66.67%; 8/12). ASCL1 upregulation in progressive astrocytoma is accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1, and increased levels of Notch ligand Delta1, which is capable of inhibiting Notch signaling by forming intracellular Notch ligand autonomous complexes. Our results imply that inhibition of Notch signaling may be an important early event in the development of grade II DA and subsequent progression to grade III AA and secondary GBM. Furthermore, ASCL1 appears to be a putative marker to distinguish primary GBM from secondary GBM.