Development of TaqMan PCR assay for genotyping SNP rs211250281 of the bovine agpat6 gene.

Milk fat percentage is an important production trait of dairy cattle and is one of the goals of breeding programs. Over 95% of the milk fat accounts for triacylglycerols. AGPAT6 (1-acylglycerol-3-phosphate O-acyltransferase 6) catalyzes an intermediary step of triglyceride synthesis in the mammary cells. Genome-wide association studies identified SNP rs211250281 (g27: 36520069 G/T) in the agpat6 gene associated with milk fat content and fat-to-protein ratio in dairy cattle. The article presents data on the development of TaqMan PCR assay for genotyping SNP rs211250281 of the bovine agpat6 gene. In this method, a primer pair, initiating amplification of 75-bp fragments of the agpat6 gene, and two allele-specific TaqMan probes are used. Identification of the G and T alleles is based on a comparison of the final fluorescence intensity of FAM and VIC dyes, respectively. The developed TaqMan PCR assay was validated by Sanger sequencing method. The results of both methods fully coincided, that confirmed high accuracy of the developed TaqMan PCR assay. This reliable, simple, rapid, and high-throughput method could be a suitable tool for studying the distribution of the SNP rs211250281 among different cattle breeds and its association with milk fat content.

Identification of novel integration sites for bovine leukemia virus proviral DNA in cancer driver genes in cattle with persistent lymphocytosis.

Enzootic bovine leukosis is one of the unsolved problems of cattle breeding in many countries. The etiological agent of the disease is the bovine leukemia virus (BLV) - an oncogenic retrovirus, that infects B-lymphocytes in cattle. The number and genetic content of BLV provirus integration sites in the bovine genome were reported to can be used as an early diagnostic sign of leukemogenesis in the infected cattle, but patterns of BLV provirus integration into the bovine genome and associations between genomic features of the integration sites and development of lymphocytosis and B-cell lymphomas remain poorly elucidated. Here we present data on five novel BLV provirus integration sites in the genome of cattle with persistent lymphocytosis. Two of these sites were located in introns of scfd2 and pgpep1 genes, which have been recognized as cancer driver genes. Three of the rest integration sites were found in the intergenic spaces between ctps1 and cited4, nampt and ccdc71, skp2 and lmbrd2 genes, from which cited4 and skp2 also possess oncogenic properties. These data support previous findings of the association between localization of BLV proviral DNA near cancer driver genes and leukemogenesis in the BLV-infected cattle.

Development of TaqMan PCR assay for detection of A and B variants of the bovine ß-lactoglobulin.

ß-Lactoglobulin (BLG) is one of the prevalent whey protein in cattle. To date, several variants of bovine BLG have been found, but the most common are A and B, which differ from each other by SNPs rs109625649 and rs110066229. Numerous studies showed effects of A and B variants of BLG on milk yield, fat and protein content and cheese-making properties. To date, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (ASPCR), PCR single-strand conformation polymorphism (PCR-SSCP) and high resolution melting (HRM) methods have been proposed for detection of A and B variants of bovine BLG. These methods involve multistep sample processing, which is an essential disadvantage in conducting large-scale cattle genotyping projects. This article describes a development of TaqMan PCR assay for detection of A and B variants (rs109625649) of bovine BLG. In this method a primer pair, initiating amplification of 101-bp fragment of BLG gene, and two allele-specific TaqMan probes are used. Identification of B and A variants of BLG is based on comparison of final fluorescence intensity of FAM and VIC dyes, respectively. The developed one-step method requires less time and is more suitable for large-scale genotyping of cattle compared to the commonly used PCR-RFLP.

Changes in Brain Responses to Music and Non-music Sounds Following Creativity Training Within the "Different Hearing" Program.

The "Different Hearing" program (DHP) is an educational activity aimed at stimulating musical creativity of children and adults by group composing in the classroom, alternative to the mainstream model of music education in Czechia. Composing in the classroom in the DHP context does not use traditional musical instruments or notation, instead, the participants use their bodies, sounds originating from common objects as well as environmental sounds as the "elements" for music composition by the participants' team, with the teacher initiating and then participating and coordinating the creative process, which ends with writing down a graphical score and then performing the composition in front of an audience. The DHP methodology works with a wide definition of musical composition. We hypothesized that the DHP short-term (2 days) intense workshop would induce changes in subjective appreciation of different classes of music and sound (including typical samples of music composed in the DHP course), as well as plastic changes of the brain systems engaged in creative thinking and music perception, in their response to diverse auditory stimuli. In our study, 22 healthy university students participated in the workshop over 2 days and underwent fMRI examinations before and after the workshop, meanwhile 24 students were also scanned twice as a control group. During fMRI, each subject was listening to musical and non-musical sound samples, indicating their esthetic impression with a button press after each sample. As a result, participants' favorable feelings toward non-musical sound samples were significantly increased only in the active group. fMRI data analyzed using ANOVA with post hoc ROI analysis showed significant group-by-time interaction (opposing trends in the two groups) in the bilateral posterior cingulate cortex/precuneus, which are functional hubs of the default mode network (DMN) and in parts of the executive, motor, and auditory networks. The findings suggest that DHP training modified the behavioral and brain response to diverse sound samples, differentially changing the engagement of functional networks known to be related to creative thinking, namely, increasing DMN activation and decreasing activation of the executive network.

Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood.

Bovine anaplasmosis is a tick-borne rickettsial disease, causing significant economic losses in many countries. The main causative agent of bovine anaplasmosis is Anaplasma marginale (Rickettsiales, Anaplasmataceae). To date, several PCR assays for A. marginale DNA detection were proposed, but most of them do not provide an internal amplification control, which allows to prevent false-negative results and is required for reliability of the results of pathogen DNA detection by PCR assay. In the present study, a real-time PCR assay based on the species-specific and highly conserved fragment of msp1α gene was developed for detection and quantification of A. marginale in bovine blood. The real-time PCR assay is able to detect as few as one copу of msp1α gene per reaction. To prevent false-negative results, simultaneous amplification and detection of the bovine genomic DNA fragment as an endogenous internal amplification control (IAC) was provided. The assay can be used as a highly specific and sensitive method for detection and quantification of A. marginale in infected cattle, and for the evaluation of the efficacy of anti-rickettsial drugs and anaplasmosis vaccines.

Molecular survey and genetic characterization of Anaplasma marginale isolates in cattle from two regions of Russia.

Anaplasma marginale is an intraerythrocytic tick-borne rickettsial pathogen that causes bovine anaplasmosis, an economically important disease of cattle worldwide. Major surface protein MSP1α has been used as a stable marker in identifying geographical strains of A. marginale. The genetic diversity of A. marginale based on MSP1α has been reported in several countries all over the world. Only a few molecular surveys of A. marginale strains have been conducted in Russia. The aim of this study was molecular detection and characterization of A. marginale isolates in cattle from two regions of Russia. Blood samples from 62 cattle were collected and screened for the presence of A. marginale by real-time PCR targeting the msp4 gene. Anaplasma marginale DNA was detected in 26 cattle (42%). The partial msp1α gene containing tandem repeat sequences and msp4 gene were amplified from msp4-positive samples, cloned and sequenced. Sequence analysis revealed that two msp4 genotypes were found. The genetic diversity of A. marginale strains was analyzed based on the MSP1α tandem repeats structure and 5'-UTR microsatellite. Sixteen new genotypes of A. marginale were found in 17 animals. Seven animals (41%) were infected by more than one genotype. Eight new tandem repeats are described for the first time. The number of repeats differed between 1 and 6 across the isolates. The msp1α microsatellite analysis revealed that six genotypes were identified; one of them was not previously described. Phylogenetic analysis revealed that Russian isolates formed four separate clades. The tandem repeat and microsatellite analyses of the msp1α gene showed a high genetic diversity among the isolates. The present study provided the first evidence of genetic diversity of A. marginale in cattle in Russia.

Impact of mash and crumble diets on intestinal amino acids transporters, intestinal morphology and pancreatic enzyme activity of broilers.

The objective of this study was to evaluate effects of mash and crumble pre-starter diets on pancreatic enzyme activity, intestinal morphology, gene expression of intestinal peptide and amino acid (AA) transporters of broilers. Broilers in battery cages were assigned to different feed forms of pre-starter diet from 1 to 10 days of age. Significantly increased body weight gain (BWG), feed intake (FI) and lowered FCR were observed in birds fed crumble pre-starter diet (CPD, p < 0.05). Feed forms had no effect on whole and small intestine length, but relative intestinal length and relative small intestinal length significantly increased in the broilers fed a mash pre-starter diet (MPD, p < 0.05). Feeding CPD increased the weight of pancreas (p < 0.05), but relative weight of the pancreas was not influenced by treatments. Pancreatic protease and amylase activities significantly increased in the broilers fed CPD (p < 0.05) but the activity of lipase was not influenced. Crypt depth (CD) and villus height (VH) were higher in broilers fed CPD (p < 0.05) but villus width (VW), villus surface area (VSA) and villus height-to-crypt depth ratio (VCR) were not influenced by treatments. mRNA levels for peptide transporter 1 (PepT1), Na+ -independent cationic AA transporter1 (CAT1), Na+ -independent cationic and Na+ -dependent neutral AA transporter 1 (y+ LAT1) and Na+ -dependent neutral AA transporter (B0 AT) were lower in birds fed CPD (p < 0.05). There were no differences in mRNA abundance of Na+ -independent cationic and zwitterionic AA transporter (b0,+ AT) among treatments. Overall, the present data showed that feeding crumble diet during first 10 days of age, through higher FI, enhanced intestinal histomorphology, increased digestive enzyme activity is beneficial to growth performance of broilers. Indeed, dietary form can be an important factor in the expression of jejunal transporters.

Intestinal amino acid and peptide transporters in broiler are modulated by dietary amino acids and protein.

This study evaluated the effect of three levels of digestible amino acids (DAA; 100, 107 and 114% of Cobb recommendations) on mRNA abundance of peptide (PepT1) and amino acid (AA) transporters in 480-day-old broilers during prestarter period. Jejunal mRNA levels of the PepT1 and b0,+AT increased as DAA level increased from 100 to 114%. The expression of CAT1 mRNA in the jejunum was higher in birds fed 100% DAA diet. The transport systems B0AT and y+LAT1 were not affected by the dietary treatments. These results demonstrated that dietary content of protein and DAA differentially affected the expression of intestinal peptide and AA transporters to modulate absorption of peptide and AA in broilers.

Sorbents with non-covalently immobilized ß-diketones for preconcentration of rare earth elements.

A comparison of the efficiency of sorbents obtained by different methods of non-covalent immobilization of ß-diketones on some low-polar matrices with respect to extraction of rare earth elements (REEs) was carried out. It was shown that sorbents containing reagent amounts of 1-8mmol/g can be obtained by sorption of reagents on low-polar matrices from aqueous and aqueous-organic solutions, and the value for the maximum capacity of the sorbent correlates with the specific surface of the matrix. Similar sorbents were also prepared by impregnating the matrix with reagent. It was found out that, under the chosen conditions, sorbents modified by extracting reagent from the aqueous solutions are more stable and extract lanthanum with higher distribution coefficients than those obtained by impregnation. We have found conditions for quantitative extraction of REEs from seawater in the proposed preconcentration systems (pH 4.0, minicolumn dimensions 2×10mm, v=4ml/min). It was shown that all REE may be quantitatively recovered in both ways: on modified sorbents and as complexes with reagents on unmodified matrices. We have proposed a sorbent for lanthanum preconcentration from large volumes of water samples (500ml). The sorbent is stable in dynamic conditions and is based on hyper cross-linked polystyrene modified with 1-phenyl-3-methyl-4-benzoylpyrazol-5-one (PMBP). Desorption could be carried out with 1-2M HNO3. REEs were determined by ICP-MS, LODs achieved were in ng/l range.