Analysis of 22 mutations within milk protein genes in Italian Friesian cattle

The bovine milk protein caseins, αS1-CN, β-CN, αS2-CN, and κ-CN are codified by four well characterized genes, named CSN1S1, CSN2, CSN1S2, and CSN3 respectively and clustered in a region of 250-kb of chromosome 6. A recent revision of milk protein nomenclature considering only protein polymorphisms indicates 8 αS1-CN, 4 αS2-CN, 12 β-CN, and 11 κ-CN variants within the genus Bos. Other mutations were found in the non-coding regions of the cluster, such as the promoter regions or the 3'UTR. Many of these polymorphisms, together with others in various genes, such as the one coding for β-lactoglobulin (LGB), show important associations with different milk quality traits. Analyzing all these polymorphisms could help clarify the role of both the casein haplotype and the other polymorphisms in milk composition and cheese-making properties, and could explain which polymorphisms are really or mostly involved. The mPCR-LDR-UA approach recently developed to test simultaneously 22 SNPs in DNA regions responsible for milk protein expression was used to type 250 Italian Friesian cattle. In perfect agreement with literature, the most frequent alleles were CSN1S1*B, CSN2*A2, CSN3*A, variant 2 of CSN1S1 promoter, and variant A of Bov-A2 element. A quite balanced frequency was observed for the LGB*A and LGB*B. No CSN2*C, CSN3*C, and CSN3*H alleles were found. The CSN1S1*C, CSN2*A3, CSN2*I alleles were detected only at the heterozygous condition and at a frequency lower than 2%. The method allowed also finding some unusual intragenic haplotype, such as the Bov-A2 element-CSN3 haplotypes A-B and B-E. As to LGB one of the four SNPs tested was always homozygous for the same mutation, as already noticed. This finding confirms that this synonymous SNP is probably a sequencing mistake or a rare mutation not decisive for the LGB typing in the Italian Friesian. Reducing cost and time for typing simultaneously many SNPs, the method will be applied to a greater number of individuals and to other breeds, aiming to find out a number of animals for each haplotype sufficient for accurate statistical analysis to give a better understanding of the significance of milk protein polymorphism

Genome-wide population structure, homozygosity, and heterozygosity patterns of Nero Siciliano pig in the framework of Italian and cosmopolitan breeds

Analysis of genomic data is becoming more and more common for the effective management of livestock breeding programmes, even in the case of local populations. In this work, the genome-wide data of Nero Siciliano pig breed were compared to that of wild boar, Italian local and cosmopolitan breeds to investigate its genetic structure, and runs of homozygosity (ROH) and heterozygosity patterns. The Nero Siciliano has been reported to have the highest rate of genetic diversity among the Italian breeds, and a genetic variability comparable to that of the cosmopolitan breeds. Analyses of genomic structure and relationships underlined its proximity to wild boar, and an internal substructure probably linked to different family lines. The breed showed a low value of inbreeding estimated from ROH, and the highest diversity index among the Italian breeds, even if lower than that of the cosmopolitans. Four ROH islands in three chromosomes (SSC8, SSC11, and SSC14) and one heterozygosity-rich region (SSC1) were identified in Nero Siciliano, highlighting genomic regions related to productive QTL. Across breeds, SSC8 and SSC14 were the chromosomes with most ROH islands, with Mora Romagnola and wild boar showing the highest level of autozygosity. Chromosomes SSC2, SSC6, SSC8 and SSC13 showed the majority of runs of heterozygosity regions, mainly found in the cosmopolitan pig breeds, which reported several genes associated with health-related QTL. The outlined results can help to better identify the genomic profile of this local breed in order to plan matings, maintain adequate internal diversity and exploit the production system

A first glance on the epigenome of Capra hircus

DNA methylation and microRNAs (miRNA) are two important forms of epigenetic modifications that play an important role in gene regulation in animals. Methylation at the carbon 5 position of cytosine residues is a fundamental layer of cellular differentiation through the control of transcriptional potential. MiRNA are small noncoding RNA molecules that regulate gene expression. Complete DNA methylomes for several organisms are now available; at the present, methylome of the domestic goat is unexplored. There is also still limited knowledge about miRNAs expression profiles in small ruminant species. Therefore, to contribute information on epigenetic modification in Capra hircus, we analysed the methylome and the miRNA population of three tissues (hypothalamus, pituitary and ovary) from 3 adult Saanen goats. We used Methylated DNA binding domain sequencing with enrichment of methylated DNA fragments and next generation sequencing. We produced least 23 million reads per sample, which were aligned to the goat reference genome. Further analyses were performed to identify peaks corresponding to hyper-methylated regions. We sequenced miRNAs expressed in the three tissues with Illumina high-throughput sequencing. Reads were mapped on the Capra hircus reference genome and both known and novel miRNAs, and miRNA target sites were identified using information collected in miRBase and using specific bioinformatic tools. This study produced a comprehensive miRNA profile related to the biology of goat. Furthermore, this is the first work dealing with methylome in Capra hircus: our preliminary results could provide new information for a deeper comprehension of epigenetic mechanisms of this species

The methylome of the hypothalamus of prepubertal and pubertal goats

Puberty is the fulfillment of fertility, a process involving physiological and morphological development. It is well known that the increased hypothalamic secretion of the gonadotropin-releasing hormone (GnRH) is essential for the activation of this process, even if the elements coordinating the timing of puberty have not been fully identified1,2. Recent studies provide proof that there is an epigenetic regulation of female puberty, and DNA methylation, the most studied epigenetic modification, plays a major role in it3. We analyzed DNA methylation patterns of 5 Alpine goats at their prepubertal stage and 5 that reached puberty in order to highlight differences in their methylome. Detection of methylated regions across the goat genome involved a Methyl Binding Domain (MBD) enrichment followed by deep sequencing (Hiseq2000 Illumina). The software ChIPseeqer4 permitted the identification of peaks corresponding to hyper-methylated regions. We have observed a higher methylation level in prepubertal goats. The distribution of the methylation peaks across the genome and within CpG islands per chromosome per group of animals has been analyzed. Furthermore, we have investigated differential methylation in genes associated with puberty. Specifically, Cbx7, coding for a core component of the Polycomb group silencing complex, and GnRHR, the gene coding for GnRH receptor, showed a higher number of peaks into two intragenic fragments within prepubertal goats. These results, accompanied by transcriptome analysis, provide a foundation for elucidating the role of DNA methylation in the complex mechanisms that drive puberty in goat species

Generation of the Becker muscular dystrophy patient derived induced pluripotent stem cell line carrying the DMD splicing mutation c.1705-8 T>C

Becker Muscular dystrophy (BMD) is an X-linked syndrome characterized by progressive muscle weakness. BMD is generally less severe than Duchenne Muscular Dystrophy. BMD is caused by mutations in the dystrophin gene that normally give rise to the production of a truncated but partially functional dystrophin protein. We generated an induced pluripotent cell line from dermal fibroblasts of a BMD patient carrying a splice mutation in the dystrophin gene (c.1705-8 T>C). The iPSC cell-line displayed the characteristic pluripotent-like morphology, expressed pluripotency markers, differentiated into cells of the three germ layers and had a normal karyotype

Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells

White lupin (Lupinus albus, L.), a widely cultivated crop that has been consumed for many years in Western Europe, may provide a useful alternative for individuals wishing to substitute animal with plant proteins for cardiovascular disease prevention. Lupin seeds have a very low content of isoflavones, and lupin protein isolates are essentially isoflavone free. In rats fed a casein-based cholesterol + cholic acid diet, a relatively low daily intake (50 mg/d by gavage for 2 wk) of total lupin protein extract reduced plasma total and VLDL + LDL cholesterol concentrations by 21 and 30%, respectively (both P<0.001). In an attempt to elucidate the lipid-lowering mechanism, LDL receptor activity was evaluated in a human hepatoma cell line (HepG2). In this model, the lupin total protein extract was essentially inactive, whereas one purified minor protein component, conglutin gamma, had a remarkable upregulatory effect, with maximal increases of 53 and 21% (both P<0.05) for LDL uptake and degradation, respectively. This initial study indicates that lupin, although isoflavone free, has hypocholesterolemic activity similar to that of other leguminous proteins in an established animal model. Further, the cholesterol reduction appears to be associated with stimulation of LDL receptors by a well-defined protein component of the lupin seeds as demonstrated by in vitro studies

Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse

BACKGROUND: Cocaine use seems to be increasing in some urban areas worldwide, but it is not straightforward to determine the real extent of this phenomenon. Trends in drug abuse are currently estimated indirectly, mainly by large-scale social, medical, and crime statistics that may be biased or too generic. We thus tested a more direct approach based on 'field' evidence of cocaine use by the general population. METHODS: Cocaine and its main urinary metabolite (benzoylecgonine, BE) were measured by mass spectrometry in water samples collected from the River Po and urban waste water treatment plants of medium-size Italian cities. Drug concentration, water flow rate, and population at each site were used to estimate local cocaine consumption. RESULTS: We showed that cocaine and BE are present, and measurable, in surface waters of populated areas. The largest Italian river, the Po, with a five-million people catchment basin, steadily carried the equivalent of about 4 kg cocaine per day. This would imply an average daily use of at least 27 ± 5 doses (100 mg each) for every 1000 young adults, an estimate that greatly exceeds official national figures. Data from waste water treatment plants serving medium-size Italian cities were consistent with this figure. CONCLUSION: This paper shows for the first time that an illicit drug, cocaine, is present in the aquatic environment, namely untreated urban waste water and a major river. We used environmental cocaine levels for estimating collective consumption of the drug, an approach with the unique potential ability to monitor local drug abuse trends in real time, while preserving the anonymity of individuals. The method tested here – in principle extendable to other drugs of abuse – might be further refined to become a standardized, objective tool for monitoring drug abuse

Lay People Representations on the Common Good and Its Financial Provision

The financial contribution to the common good is a relevant issue to contemporary societies, especially in the wake of the Global Financial Crisis. In the economic literature, taxes and monetary donations have been regarded as two complementary ways of financially providing for the common good. In the psychological literature, instead, they have not been studied in conjunction. In-depth interviews have been conducted using interpretative phenomenological analysis (IPA) approach and a photo-elicitation technique to investigate the representations people share on the financial provision for the common good. Results suggest that both taxes and donations are seen as indirect, rather than direct, ways of providing for the common good. From a formal and cognitive level, paying taxes and making donations can be seen as two sides of the same coin, but they present differences at the affective level. When paying taxes, people are concerned mostly about the effects and expect a material exchange in return; when making a monetary donation, people are concerned mostly about the motivations and expect an emotional exchange in return