Milena Kovač

Genome-wide and species-wide in silico screening for intragenic MicroRNAs in human, mouse and chicken.

MicroRNAs (miRNAs) are non-coding RNAs (ncRNAs) involved in regulation of gene expression. Intragenic miRNAs, especially those exhibiting a high degree of evolutionary conservation, have been shown to be coordinately regulated and/or expressed with their host genes, either with synergistic or antagonistic correlation patterns. However, the degree of cross-species conservation of miRNA/host gene co-location is not known and co-expression information is incomplete and fragmented among several studies. Using the genomic resources (miRBase and Ensembl) we performed a genome-wide in silico screening (GWISS) for miRNA/host gene pairs in three well-annotated vertebrate species: human, mouse, and chicken. Approximately half of currently annotated miRNA genes resided within host genes: 53.0% (849/1,600) in human, 48.8% (418/855) in mouse, and 42.0% (210/499) in chicken, which we present in a central publicly available Catalog of intragenic miRNAs (http://www.integratomics-time.com/miR-host/catalog). The miRNA genes resided within either protein-coding or ncRNA genes, which include long intergenic ncRNAs (lincRNAs) and small nucleolar RNAs (snoRNAs). Twenty-seven miRNA genes were found to be located within the same host genes in all three species and the data integration from literature and databases showed that most (26/27) have been found to be co-expressed. Particularly interesting are miRNA genes located within genes encoding for miRNA silencing machinery (DGCR8, DICER1, and SND1 in human and Cnot3, Gdcr8, Eif4e, Tnrc6b, and Xpo5 in mouse). We furthermore discuss a potential for phenotype misattribution of miRNA host gene polymorphism or gene modification studies due to possible collateral effects on miRNAs hosted within them. In conclusion, the catalog of intragenic miRNAs and identified 27 miRNA/host gene pairs with cross-species conserved co-location, co-expression, and potential co-regulation, provide excellent candidates for further functional annotation of intragenic miRNAs in health and disease.

Obesity gene atlas in mammals.

Obesity in humans has increased at an alarming rate over the past two decades and has become one of the leading public health problems worldwide. Studies have revealed a large number of genes/markers that are associated with obesity and/or obesity-related phenotypes, indicating an urgent need to develop a central database for helping the community understand the genetic complexity of obesity. In the present study, we collected a total of 1,736 obesity associated loci and created a freely available obesity database, including 1,515 protein-coding genes and 221 microRNAs (miRNAs) collected from four mammalian species: human, cattle, rat, and mouse. These loci were integrated as orthologs on comparative genomic views in human, cattle, and mouse. The database and genomic views are freely available online at: http://www.integratomics-time.com/fat_deposition. Bioinformatics analyses of the collected data revealed some potential novel obesity related molecular markers which represent focal points for testing more targeted hypotheses and designing experiments for further studies. We believe that this centralized database on obesity and adipogenesis will facilitate development of comparative systems biology approaches to address this important health issue in human and their potential applications in animals.

Genome-wide in silico screening for microRNA genetic variability in livestock species

MicroRNAs are a class of non-coding RNAs that post-transcriptionally regulate target gene expression. Previous studies have shown that microRNA gene variability can interfere with its function, resulting in phenotypic variation. Polymorphisms within microRNA genes present a source of novel biomarkers for phenotypic traits in animal breeding. However, little is known about microRNA genetic variability in livestock species, which is also due to incomplete data in genomic resource databases. Therefore, the aim of this study was to perform a genome-wide in silico screening of genomic sources and determine the genetic variability of microRNA genes in livestock species using mirna sniper 3.0 (http://www.integratomics-time.com/miRNA-SNiPer/), a new version of our previously developed tool. By examining Ensembl and miRBase genome builds, it was possible to design a tool-based generated search of 16 genomes including four livestock species: pig, horse, cattle and chicken. The analysis revealed 65 polymorphisms located within mature microRNA regions in these four species, including 28% within the seed region in cattle and chicken. Polymorphic microRNA genes in cattle and chicken were further examined for mapping to quantitative trait loci regions associated with production and health traits. The developed bioinformatics tool enables the analysis of polymorphic microRNA genes and prioritization of potential regulatory polymorphisms and therefore contributes to the development of microRNA-based biomarkers in livestock species. The assembled catalog and the developed tool can serve the animal science community to efficiently select microRNA SNPs for further quantitative and molecular genetic evaluations of their phenotypic effects and causal associations with livestock production traits.

Comparison of piecewise Weibull baseline survival models for estimation of true and functional longevity in Brown cattle raised in small herds

Piecewise Weibull proportional hazard models were used to investigate the effect of genetic and nongenetic factors on functional and true longevity traits of the Slovenian Brown cattle breed. Records of 37 908 Brown cows from 2401 Slovenian herds were used. As these herds were characterised by a relatively small average herd size starting from 6.7 in 1999 and increasing to 8.7 Brown cows per herd in 2008, milk yield classification was made within different herd size groups. The hazard rate was the lowest in the first part of each lactation and was increasing for later stages. Culling risk was lower for cows from herds increasing in size, for cows with higher milk production and for cows from a region with smaller herd sizes and tougher conditions for cattle breeding. The latter result is surprising and may be related to better attention to maintain the animals, despite their lower milk production. The introduction of the milk quota system and drought was found to have an important effect on culling policy between the last seasons of the years 2001 and 2003. Seasonal effects were not related to the milk quota year (from April to March), but to the effect of shortage in fodder during the winter time. The effect of age at first calving and the interaction between year and milk yield class were not found to be significant. Heritability for functional and for true longevity were similar at around 10% each. Inclusion of a correction for class of milk yield to approximate functional longevity increased the herd-year random effect variance by 53%, whereas the sire variance increased by only 14%. The correlation coefficient between ranks of breeding values for functional and true longevity was high (0.91), whereas genetic trends were not found to be significant. To assess their predictive ability, models were compared looking at the survival rate of 4212 second-crop daughters not included in the initial models. The average correlation between estimated breeding values and survival at different stages was 0.39 for true longevity and 0.43 for functional longevity. Results showed that ranking milk yield at population level is appropriate to correct for voluntary culling on low production in small herds.

Effect of sex and slaughter weight on meat and fat quality of the Krškopolje pig reared in an enriched environment

Improvements in meat quality are sought through sources of variation along the meat chain, including breed and production system. Hence, the aim of this study was to determine slaughter weight and sex effects on the meat quality of the indigenous Krskopolje pig breed reared in a feed-enriched indoor environment for the growing-finishing periods. Raised in this enriched environment, the intramuscular fat (IMF) content of the loin was 1.5–2.5% higher than previously reported for other production systems. Slaughter weight (119–132 kg or >135–170 kg) influences on meat colour and IMF composition were observed. Animals from the light group had lighter coloured meat with lower Japanese colour scores as well as higher saturated fatty acid (SFA) and lower monounsaturated fatty acid (MUFA) proportions in the IMF. A lower proportion of MUFA was also observed in the IMF from gilts than barrows. Furthermore, a higher proportion of SFA was found in the IMF from the gilts. However, the lower IMF content in the gilts negates any apparent compositional advantages between the sexes. The IMF content and composition of the Krskopolje pig reared in an enriched environment suggests good commercial potential of this breed.

Comparison between sire-maternal grandsire and animal models for genetic evaluation of longevity in a dairy cattle population with small herds

Survival analysis techniques for sire-maternal grandsire (MGS) and animal models were used to test the genetic evaluation of longevity in a Slovenian Brown cattle population characterized by small herds. Three genetic models were compared: a sire-MGS model for bulls and an approximate animal model based on estimated breeding values (EBV) from the sire-MGS model for cows, an animal model, and an animal model based on the estimated variance components from the sire-MGS model. In addition, modeling the contemporary group effect was defined as either a herd or a herd-year (HY) effect. With various restrictions on the minimum HY group size (from 1 to 10 cows per HY), changes in estimates of variance components, and consequently also in EBV, were observed for the sire-MGS and animal models. Variance of contemporary group effects decreased when an HY effect was fitted instead of a herd effect. In the case of a sire-MGS model, estimates of additive genetic variance were mostly robust to changes in minimum HY group size or fitting herd or HY effect, whereas they increased in the animal model when HY instead of herd effects was fitted, possibly revealing some confounding between cow EBV and contemporary group effect. Estimated heritabilities from sire-MGS models were between 0.091 and 0.119 and were mainly influenced by the restriction on the HY group size. Estimated heritabilities from animal models were higher: between 0.125 and 0.160 when herd effect was fitted and between 0.171 and 0.210 when HY effect was fitted. Rank correlations between the animal model and the approximate animal model based on EBV from the sire-MGS model were high: 0.94 for cows and 0.93 for sires when a herd effect was fitted and 0.90 for cows and 0.93 for sires when an HY effect was fitted. Validation performed on the independent validation data set revealed that the correlation between sire EBV and daughter survival were slightly higher with the approximate animal model based on EBV from the sire-MGS model compared with the animal model. The correlations between the sire EBV and daughter survival were higher when the model included an HY effect instead of a herd effect. To avoid confounding and reduce computational requirements, it is suggested that the approximate animal model based on EBV from the sire-MGS model and HY as a contemporary group effect is an interesting compromise for practical applications of genetic evaluation of longevity in cattle populations.

Assessment of differential expression of oncogenes in adenocarcinoma of stomach with fluorescent labeling and simultaneous amplification of gene transcripts

Assessment of differential expression of oncogenes in adenocarcinoma of stomach with fluorescent labeling and simultaneous amplification of gene transcripts Background. Gastric cancer is one of the leading malignancies with a poor prognosis and low survival rates. Although the mechanisms underlying its development are still unknown, there is a consensus that genetic instability, inactivation of tumor suppressor genes and over-expression of oncogenes are involved in the early and late stages of gastric carcinogenesis. In the present study we wanted to display differential expression of seven oncogenes, namely CCNE1, EGF, ERBB3, FGF4, HRG1, HGFR and TDGF1. Patients and methods. We employed a method based on the multiplex reverse transcription polymerase chain (RT-PCR) method with a fluorescence detection. Results. More than half of patients (74.3%) out of total 74 with gastric adenocarcinoma had over-expressed at least one oncogene, with the exception of FGF4, which was expressed in tumor tissue of less than one third of patients. 56.8% of the patients patients showed over-expression of two or more oncogenes. Conclusions. Patients with precancerous lesions had elevated levels of TDGF1 or cripto-1 (64.9%) and CCNE1 (57.1%), suggesting that they could be used as markers for an early detection of malignant changes in stomach. Finally, the fluorescent multiplex RT-PCR method could be of value for rapid assessment of oncogene mRNA levels in small samples of tumor or precancerous biopsies.

FTO gene variants are associated with growth and carcass traits in cattle.

An important aim in animal breeding is the improvement of growth and meat quality traits. Previous studies have demonstrated that genetic variants in the fat mass and obesity associated (FTO) gene have a relatively large effect on human obesity as well as on body composition in rodents and, more recently, in livestock. Here, we examined the effects of the FTO gene variants on growth and carcass traits in the Slovenian population of Simmental (SS) and Brown (SB) cattle. To validate and identify new polymorphisms, we used sequencing, PCR-RFLP analysis and TaqMan assays in the SS breed and FTO gene variants data from the Illumina BovineSNP50 v1 array for the SB breed. Sequencing of the eight samples of progeny-tested SS sires detected 108 single nucleotide polymorphisms (SNPs) in the bovine FTO gene. Statistical analyses between growth and carcass traits and 34 FTO polymorphisms revealed significant association of FTO variants with lean meat percentage in both breeds. Additionally, FTO SNPs analyzed in SS cattle were associated with fat percentage, bone weight and live weight at slaughter. The FTO gene can thus be regarded as a candidate gene for the marker-assisted selection programs in our and possibly other populations of cattle. Future studies in cattle might reveal novel roles for the FTO gene in shaping carcass traits in livestock species as well as body composition control in other mammals.

Nutritional quality of Krškopolje and commercial fattener pig meats in Slovenia.

Abstract The nutritional quality of M. Longissimus dorsi in the autochthonous Krškopolje pig breed and commercial fatteners from intensive Slovenian farms was compared. Commercial fatteners were divided into two groups according to lean meat percentage: MEATY and FATTY groups. The Krškopolje pigs (KK group) and the FATTY group had higher intramuscular fat content than the MEATY group. The lowest saturated fatty acids (SFA) proportion was found in the KK group and the highest in the FATTY group. The KK and FATTY groups contained higher proportions of monounsaturated fatty acids (MUFA) as well as lower proportions of polyunsaturated fatty acids (PUFA) compared to the MEATY group. The highest n-6 PUFA proportion was found in the MEATY group. The n-3 PUFA proportion differed between the MEATY and FATTY groups. The FATTY group had the lowest ratio of polyunsaturated to saturated fatty acids as well as the highest atherogenic index.