Stereo-Specific Modulation of the Extracellular Calcium-Sensing Receptor in Colon Cancer Cells

Pharmacological allosteric agonists (calcimimetics) of the extracellular calcium-sensing receptor (CaSR) have substantial gastro-intestinal side effects and induce the expression of inflammatory markers in colon cancer cells. Here, we compared the effects of both CaSR-specific (R enantiomers) and -unspecific (S enantiomers) enantiomers of a calcimimetic (NPS 568) and a calcilytic (allosteric CaSR antagonists; NPS 2143) to prove that these effects are indeed mediated via the CaSR, rather than via off-target effects, e.g., on β-adrenoceptors or calcium channels, of these drugs. The unspecific S enantiomer of NPS 2143 and NPS S-2143 was prepared using synthetic chemistry and characterized using crystallography. NPS S-2143 was then tested in HEK-293 cells stably transfected with the human CaSR (HEK-CaSR), where it did not inhibit CaSR-mediated intracellular Ca2+ signals, as expected. HT29 colon cancer cells transfected with the CaSR were treated with both enantiomers of NPS 568 and NPS 2143 alone or in combination, and the expression of CaSR and the pro-inflammatory cytokine interleukin 8 (IL-8) was measured by RT-qPCR and ELISA. Only the CaSR-selective enantiomers of the calcimimetic NPS 568 and NPS 2143 were able to modulate CaSR and IL-8 expression. We proved that pro-inflammatory effects in colon cancer cells are indeed mediated through CaSR activation. The non-CaSR selective enantiomer NPS S-2143 will be a valuable tool for investigations in CaSR-mediated processes

Design and validation of a 90K SNP genotyping assay for the water buffalo (Bubalus bubalis)

Background: The availability of the bovine genome sequence and SNP panels has improved various genomic analyses, from exploring genetic diversity to aiding genetic selection. However, few of the SNP on the bovine chips are polymorphic in buffalo, therefore a panel of single nucleotide DNA markers exclusive for buffalo was necessary for molecular genetic analyses and to develop genomic selection approaches for water buffalo. The creation of a 90K SNP panel for river buffalo and testing in a genome wide association study for milk production is described here. Methods: The genomes of 73 buffaloes of 4 different breeds were sequenced and aligned against the bovine genome, which facilitated the identification of 22 million of sequence variants among the buffalo genomes. Based on frequencies of variants within and among buffalo breeds, and their distribution across the genome, inferred from the bovine genome sequence, 90,000 putative single nucleotide polymorphisms were selected to create an Axiom® Buffalo Genotyping Array 90K. Results: This 90K "SNP-Chip" was tested in several river buffalo populations and found to have ∼70% high quality and polymorphic SNPs. Of the 90K SNPs about 24K were also found to be polymorphic in swamp buffalo. The SNP chip was used to investigate the structure of buffalo populations, and could distinguish buffalo from different farms. A Genome Wide Association Study identified genomic regions on 5 chromosomes putatively involved in milk production. Conclusion: The 90K buffalo SNP chip described here is suitable for the analysis of the genomes of river buffalo breeds, and could be used for genetic diversity studies and potentially as a starting point for genome-assisted selection programmes. This SNP Chip could also be used to analyse swamp buffalo, but many loci are not informative and creation of a revised SNP set specific for swamp buffalo would be advised.Daniela Iamartino, Ezequiel L. Nicolazzi, Curtis P. Van Tassell, James M. Reecy, Eric R. Fritz-Waters, James E. Koltes, Stefano Biffani, Tad S. Sonstegard, Steven G. Schroeder, Paolo Ajmone-Marsan, Riccardo Negrini, Rolando Pasquariello, Paola Ramelli, Angelo Coletta, José F. Garcia, Ahmad Ali, Luigi Ramunno, Gianfranco Cosenza, Denise A.A. de Oliveira, Marcela G. Drummond, Eduardo Bastianetto, Alessandro Davassi, Ali Pirani, Fiona Brew, John L. William

Haplotype Affinities Resolve a Major Component of Goat (Capra hircus) MtDNA D-Loop Diversity and Reveal Specific Features of the Sardinian Stock

Goat mtDNA haplogroup A is a poorly resolved lineage absorbing most of the overall diversity and is found in locations as distant as Eastern Asia and Southern Africa. Its phylogenetic dissection would cast light on an important portion of the spread of goat breeding. The aims of this work were 1) to provide an operational definition of meaningful mtDNA units within haplogroup A, 2) to investigate the mechanisms underlying the maintenance of diversity by considering the modes of selection operated by breeders and 3) to identify the peculiarities of Sardinian mtDNA types. We sequenced the mtDNA D-loop in a large sample of animals (1,591) which represents a non-trivial quota of the entire goat population of Sardinia. We found that Sardinia mirrors a large quota of mtDNA diversity of Western Eurasia in the number of variable sites, their mutational pattern and allele frequency. By using Bayesian analysis, a distance-based tree and a network analysis, we recognized demographically coherent groups of sequences identified by particular subsets of the variable positions. The results showed that this assignment system could be reproduced in other studies, capturing the greatest part of haplotype diversity

The Pentro horse: genetic characterization by microsatellite markers

The Pentro horse population is an autochthonous breed from a breeding area characterized by climatic and geographicpeculiarities. The horse population is interesting because of its rusticity, however, has run the risk of loosing its identitybecause of the introduction of exotic genetic material. This study presents data of the genetic characterization by meansof microsatellite markers. A total of 12 microsatellite loci were used to score 147 individuals among those with the Pentrophenotype, as well as 16 individuals representing the variable part of the population. Allele frequencies were calculatedfor each locus, with the mean number of alleles (Nall = 6.7) and the expected heterozygosity (He = 0.724). The meanobserved heterozygosity was 0.695. The obtained data were used to compare the genetic structure of the Pentro horseto six other Italian breeds. The N-J tree computed on individual genetic distances showed that 93% of Pentro horses clusteredtogether

PrP genotype frequencies and risk evaluation for scrapie in dairy sheep breeds from southern Italy

Concerns regarding scrapie in sheep breeding have increased in the last few decades. The present study was carried out in dairy sheep breeds from southern Italy. In order to find breeding animals resistant to scrapie, the PrP genes of 1,205 animals from entire flocks of dairy native Apulian Leccese and Altamurana breeds, and Sicilian Comisana breed, were analysed for polymorphisms at codons 136, 154, and 171 related to scrapie resistance/susceptibility. The Altamurana breed was considered as two populations (Alt-Cav and Alt-Cra-Zoe), based on presumed cross-breeding. A total of five alleles and ten different genotypes were found. The ARQ allele was predominant for all breeds followed by ARR, the most resistant allele to scrapie, which was highly prevalent in Comisana (50%) and in native Alt-Cav (42.4%). The VRQ allele, associated with the highest susceptibility to scrapie, was detected at not negligeable levels in allocthonous Comisana (3.5%), at a low frequency (0.2%) in native Leccese and Alt-Cra-Zoe, while it was absent in Alt-Cav. The frequencies of PrP genotypes with a very low susceptibility risk to scrapie (R1) was higher in Comisana and Alt-Cav. The most susceptible genotype, ARQ/VRQ, was found only in Comisana. Within the Altamurana breed, there were notable differences between Alt-Cav and Alt-Cra-Zoe sheep. The Alt-Cav was characterised by the absence of VRQ and AHQ alleles and by the higher frequency of the ARR/ARR genotype (18.7%). Breeding programs, mainly in endangered breeds such as Altamurana, should be conducted gradually, combining resistance to scrapie, maintenance of genetic variability, and production

Evaluation of genetic variability within PrP genotyped sheep of endangered Italian Altamurana breed

In the last few decades, there has been increased awareness of preservation and exploitation of endangered breeds for the maintenance of biodiversity, as well as the concern for diseases in sheep breeding.This study was carried out in native endangered Altamurana dairy sheep breed from Southern Italy. The Altamurana breed was considered as two populations (Alt-Cav and Alt-Cra-Zoe), based on presumed cross-breed and remarkable differences in the PrP genotypes frequencies. The genetic diversity between the two Altamurana populations (Alt-Cav and Alt-Cra-Zoe) was evaluated in comparison to three Italian dairy breeds through fourteen microsatellite markers. Both measures of genetic distance and the population structure analysis highlighted that the Alt-Cav and Alt-Cra-Zoe sheep have a particular genetic background. The estimated fixation index (FST) and the genetic Nei's distances among the populations showed a higher level of genetic differentiation for Alt-Cav than Alt-Cra-Zoe. The Bayesian clustering analysis implemented in the STRUCTURE software showed clear and distinct clusters for the two Altamurana populations, confirming the hypothesis of Alt-Cav as a genetic group well differentiated from Alt-Cra-Zoe. Alt-Cav likely can be considered as belonging to the original strain of the Altamurana breed. This findings may be used to assist the programme for conservation and selection of scrapie resistance genotypes in endangered Altamurana sheep breed

Genomic structural differences between cattle and River Buffalo identified through comparative genomic and transcriptomic analysis

Water buffalo (Bubalus bubalis L.) is an important livestock species worldwide. Like many other livestock species, water buffalo lacks high quality and continuous reference genome assembly, required for fine-scale comparative genomics studies. In this work, we present a dataset, which characterizes genomic differences between water buffalo genome and the extensively studied cattle (Bos taurus Taurus) reference genome. This data set is obtained after alignment of 14 river buffalo whole genome sequencing datasets to the cattle reference. This data set consisted of 13,444 deletion CNV regions, and 11,050 merged mobile element insertion (MEI) events within the upstream regions of annotated cattle genes. Gene expression data from cattle and buffalo were also presented for genes impacted by these regions. Public assessment of this dataset will allow for further analyses and functional annotation of genes that are potentially associated with phenotypic difference between cattle and water buffalo

New Insights on Water Buffalo Genomic Diversity and Post-Domestication Migration Routes From Medium Density SNP Chip Data

The domestic water buffalo is native to the Asian continent but through historical migrations and recent importations, nowadays has a worldwide distribution. The two types of water buffalo, i.e., river and swamp, display distinct morphological and behavioral traits, different karyotypes and also have different purposes and geographical distributions. River buffaloes from Pakistan, Iran, Turkey, Egypt, Romania, Bulgaria, Italy, Mozambique, Brazil and Colombia, and swamp buffaloes from China, Thailand, Philippines, Indonesia and Brazil were genotyped with a species-specificmedium-density 90K SNP panel. We estimated the levels of molecular diversity and described population structure, which revealed historical relationships between populations and migration events. Three distinct gene pools were identified in pure river as well as in pure swamp buffalo populations. Genomic admixture was seen in the Philippines and in Brazil, resulting from importations of animals for breed improvement. Our results were largely consistent with previous archeological, historical and molecular-based evidence for two independent domestication events for river-and swamp-type buffaloes, which occurred in the Indo-Pakistani region and close to the China/Indochina border, respectively. Based on a geographical analysis of the distribution of diversity, our evidence also indicated that the water buffalo spread out of the domestication centers followed two major divergent migration directions: river buffaloes migrated west from the Indian sub-continent while swamp buffaloes migrated from northern Indochina via an east-south-eastern route. These data suggest that the current distribution of water buffalo diversity has been shaped by the combined effects of multiple migration events occurred at different stages of the post-domestication history of the species.Fondazione Romeo ed Enrica Invernizzi, Milan, Italy; Sao Paulo Research Foundation (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP); Doctoral School on the Agro-Food System (Agrisystem) of the Universita Cattolica del Sacro Cuore (Italy); Universita Cattolica del S. Cuore; [2016/05787-7]We thank Dr. Elisa Eufemi for the help provided in screening the scientific literature and other written sources. We also thank all the members of the International Buffalo Consortium. A previous version of this manuscript appeared as a chapter of the Ph. D. thesis by Vajana (2017). This work was partially supported by the Fondazione Romeo ed Enrica Invernizzi, Milan, Italy. MMi was supported by grant 2016/05787-7, Sao Paulo Research Foundation (FAPESP). EV was supported by the Doctoral School on the Agro-Food System (Agrisystem) of the Universita Cattolica del Sacro Cuore (Italy).; Financial support to the publication of this manuscript has been provided by the Linea D. 3.1 (year 2017) of the Universita Cattolica del S. Cuore within its activities of promotion and dissemination of scientific research

Identification of candidate genes for paratuberculosis resistance in the native Italian Garfagnina goat breed

Paratuberculosis disease is a chronic bacterial disease infection of ruminants of global relevance, caused by MAP (Mycobacterium avium subsp. paratuberculosis). The present study was conducted on the Garfagnina goat breed that is an Italian native goat population registered on the Tuscan regional repertory of genetic resources at risk of extinction. Forty-eight adult goats (27 serologically positive to MAP-positive and 21 serologically negative to MAP-negative) belonging to a single flock that had experienced annual mortalities due to MAP infection were identified and genotyped with the Illumina GoatSNP60 BeadChip. Diagnosis was achieved by serological tests, as well as post-mortem examination of affected animals. A genome-wide scan was then performed on the individual marker genotypes, in an attempt to identify genomic regions associated with MAP infection disease. Nine significant markers were highlighted and they were located within, or nearby, annotated genes. Two genes found in this study encode are linked to protein kinases that are among the most important enzymes involved in the immune response to Johne’s disease, and four genes are involved in the functions of the Golgi complex