Identification and functional characterization of a novel monotreme-specific antibacterial protein expressed during lactation

Monotremes are the only oviparous mammals and exhibit a fascinating combination of reptilian and mammalian characters. They represent a component of synapsidal reproduction by laying shelled eggs which are incubated outside the mother’s body. This is accompanied by a prototherian lactation process, marking them as representatives of early mammals. The only extant monotremes are the platypus, and the short- and long- beaked echidnas, and their distributions are limited to Australia and New Guinea. Apart for a short weaning period, milk is the sole source of nutrition and protection for the hatchlings which are altricial and immunologically naive. The duration of lactation in these mammals is prolonged relative to the gestational length and period of incubation of eggs. Much of the development of monotreme young occurs in the non-sterile ex-utero environment. Therefore the role of milk in the growth, development and disease protection of the young is of significant interest. By sequencing the cDNA of cells harvested from monotreme milk, we have identified a novel monotreme- specific transcript, and the corresponding gene was designated as the EchAMP. The expression profile of this gene in various tissues revealed that it is highly expressed in milk cells. The peptides corresponding to the EchAMP protein have been identified in a sample of echidna milk In silico analysis indicated putative antimicrobial potential for the cognate protein of EchAMP. This was further confirmed by in vitro assays using a host of bacteria. Interestingly, EchAMP did not display any activity against a commensal gut floral species. These results support the hypothesis of enhancement of survival of the young by antimicrobial bioactives of mammary gland origin and thus emphasize the protective, non- nutritional role of milk in mammals.Swathi Bisana, Satish Kumar, Peggy Rismiller, Stewart C. Nicol, Christophe Lefèvre, Kevin R. Nicholas, Julie A. Shar

Identification of SNP and SSR Markers in Finger Millet Using Next Generation Sequencing Technologies

Finger millet is an important cereal crop in eastern Africa and southern India with excellent grain storage quality and unique ability to thrive in extreme environmental conditions. Since negligible attention has been paid to improving this crop to date, the current study used Next Generation Sequencing (NGS) technologies to develop both Simple Sequence Repeat (SSR) and Single Nucleotide Polymorphism (SNP) markers. Genomic DNA from cultivated finger millet genotypes KNE755 and KNE796 was sequenced using both Roche 454 and Illumina technologies. Non-organelle sequencing reads were assembled into 207 Mbp representing approximately 13% of the finger millet genome. We identified 10,327 SSRs and 23,285 non-homeologous SNPs and tested 101 of each for polymorphism across a diverse set of wild and cultivated finger millet germplasm. For the 49 polymorphic SSRs, the mean polymorphism information content (PIC) was 0.42, ranging from 0.16 to 0.77. We also validated 92 SNP markers, 80 of which were polymorphic with a mean PIC of 0.29 across 30 wild and 59 cultivated accessions. Seventy-six of the 80 SNPs were polymorphic across 30 wild germplasm with a mean PIC of 0.30 while only 22 of the SNP markers showed polymorphism among the 59 cultivated accessions with an average PIC value of 0.15. Genetic diversity analysis using the polymorphic SNP markers revealed two major clusters; one of wild and another of cultivated accessions. Detailed STRUCTURE analysis confirmed this grouping pattern and further revealed 2 sub-populations within wild E. coracana subsp. africana. Both STRUCTURE and genetic diversity analysis assisted with the correct identification of the new germplasm collections. These polymorphic SSR and SNP markers are a significant addition to the existing 82 published SSRs, especially with regard to the previously reported low polymorphism levels in finger millet. Our results also reveal an unexploited finger millet genetic resource that can be included in the regional breeding programs in order to efficiently optimize productivity

Transcriptome analysis of mammary epithelial cell gene expression reveals novel roles of the extracellular matrix

Background The unique lactation strategy of the tammar wallaby (Macropus eugeni) has been invaluable in evaluating the role of lactogenic hormones and the extracellular matrix (ECM) in the local control of mammary gland function. However molecular pathways through which hormones and ECM exert their effect on wallaby mammary gland function remain unclear. This study undertakes transcriptome analysis of wallaby mammary epithelial cells (WallMEC) following treatment with mammary ECM from two distinct stages of lactation. Methods WallMEC from MID lactation mammary glands were cultured on ECM from MID or LATE lactation and treated for 5 days with 1 μg/ml cortisol, 1 μg/ml insulin, 0.2 µg/ml prolactin, 650 pg/ml triodothyronine and 1 pg/ml estradiol to induce lactation. WallMEC RNA from triplicate ECM treatments was used to perform RNAseq. Results ECM from MID and LATE lactation differentially regulated key genes in sugar and lipid metabolism. Seven pathways including galactose metabolism, lysosome, cell adhesion molecules (CAM), p53 signaling, the complement and coagulation and Nod-like receptor signaling pathways were only significantly responsive to ECM in the presence of hormones. The raw RNA-seq data for this project are available on the NCBI Gene Expression Omnibus (GEO) browser (accession number GSE81210). Conclusions A potential role of ECM in regulation of the caloric content of milk, among other functions including apoptosis, cell proliferation and differentiation has been identified. General significance This study has used a non-eutherian lactation model to demonstrate the synergy between ECM and hormones in the local regulation of mammary function. © 2017 The Author