The equine graying with age mutation of the STX17 gene: A copy number study using droplet digital PCR reveals a new pattern.

The equine graying with age causative mutation in the syntaxin-17 gene (STX17) has been known for over a decade, but proper genotyping of this variant remains challenging due to its molecular character (4.6-kb tandem duplication). Precise information on gray mutation status is important for horse breeders and veterinarians, since gray homozygous horses are more prone to developing aggressive melanoma tumors than heterozygotes. Since recent studies have confirmed that droplet digital PCR is a valuable technique for copy number analysis, we decided to investigate whether this method can be used for accurate genotyping of the horse graying-related variant and established the copy numbers of the 4.6-kb fragment in the available cohort (n = 75) of gray and nongray horses of various breeds. Surprisingly, we found that our STX17 genotype results varied from what has been previously published, suggesting that gray phenotype is associated with the presence of six (GG) or four (Gg) copies of studied region. All the examined nongray horses (gg) have the two copies of these fragments. This new pattern and its inheritance were also confirmed by an analysis conducted for the Polish Warmblood horse family. We noted no further copy number variation in the entire tested samples set. Our study confirmed the usefulness and accuracy of droplet digital PCR for genotyping STX17 gene variant. Further studies on a broader range of materials are needed to fully understand the origin and molecular structure of the graying causative mutation in the horse STX17.

TBX3 and ASIP genotypes reveal discrepancies in officially recorded coat colors of Hucul horses.

Although only a few specific pigmentation types are allowed within the Hucul horse registry, accurate determination of particular coat colors can be uncertain due to the presence of variation in color shades and segregation of multiple dun dilution variants. Herein, we genotyped the previously identified polymorphisms within two coat color loci TBX3 (T-box 3) and ASIP (Agouti Signaling Protein) in 462 Hucul individuals and compared the genotype predicted phenotypes with observed pigmentation types provided in the Polish Horse Breeders Association database. We identified disagreement between the predicted and recorded coat color in 157 horses (34%). The most common error was misclassification of horses with the nd1/nd1 and nd1/nd2 genotypes, what may be related with the occurrence of some 'intermediate' dilution phenotypes in such individuals. We have also proven that the frequency of the dominant dun dilution allele (D) (0.30) is higher than previously predicted by available studbooks. The D allele(s) is easily 'hidden' in various phenotypic groups including dark bay and black, therefore we hypothesized that the dun dilution effect itself is not as strongly epistatic in the Hucul horse as described in other horse breeds. This may be the result of an additional genetic modifier suppressing D allele phenotypic effect.

Elemental changes of hippocampal formation occurring during postnatal brain development.

In this paper the elemental changes of rat hippocampal formation occurring during the postnatal development were examined. Three groups of animals were used in the study. These were naive Wistar rats at the age of 6-, 30- and 60-days and the chosen life periods corresponded to the neonatal period, childhood and early adulthood in humans, respectively. For the topographic and quantitative elemental analysis X-ray fluorescence microscopy was applied and the measurements were done at the FLUO beamline of ANKA. The detailed quantitative and statistical analysis was done for four areas of hippocampal formation, namely sectors 1 and 3 of the Ammon's horn (CA1 and CA3, respectively), dentate gyrus (DG) and its internal area (hilus of DG, H). The obtained results showed that among the all examined elements (P, S, K, Ca, Fe, Cu, Zn and Se), only the levels of Fe and Zn changed significantly during postnatal development of the hippocampal formation and both the elements were significantly higher in young adults comparing to the rats in neonatal period. The increased Fe areal density was found in all examined hippocampal areas whilst Zn was elevated in CA3, DG and H. In order to follow the dynamics of age-dependent elemental changes, the statistical significance of differences in their accumulation between subsequent moments of time was examined. The obtained results showed statistically relevant increase of Zn level only in the first observation period (between 6th and 30th day of life). Afterwards the areal density of the element did not change significantly. The increase of Fe areal density took place in both examined periods, however the observed changes were small and usually not statistically relevant.

Total energy of sigma-phase Fe-Cr-X (X=Co, Ni) alloys: Calculated and modeled data.

The article contains computational data of electronic structure and crystal stability of two sigma-phases, namely Fe-Cr-Co and Fe-Cr-Ni, using the Korringa-Kohn-Rostoker method (KKR) for electronic band structure calculations. Total energy values, ET , calculated for the number of ordered unit cells with various atomic concentrations and sublattice occupancies are reported. In parallel, obtained data are modelled assuming polynomial dependence of the ET -values versus sublattice occupancies. For more details, please see the article "Site occupancies in sigma-phase Fe-Cr-X (X=Co, Ni) alloys: Calculations versus experiment" (J. Cieslak, J. Tobola, S.M. Dubiel, 2016) [1].

Evaluation of the Debye temperature for iron cores in human liver ferritin and its pharmaceutical analogue, Ferrum Lek, using Mössbauer spectroscopy.

An iron-polymaltose complex, Ferrum Lek, used as antianemic drug and considered as a ferritin analogue and human liver ferritin were investigated in the temperature range of 295-90K using (57)Fe Mössbauer spectroscopy with a high velocity resolution (in 4096 channels). This study aimed to make a comparison of the Fe atom dynamics in the Ferrum Lek and ferritin iron cores by means of evaluation of the Debye temperature using the temperature dependence of the spectral center shift obtained with two different fitting procedures and the second order Doppler shift approach. The Debye temperature, evaluated as ΘD=502±24K for Ferrum Lek and ΘD=461±16K for human liver ferritin, demonstrated a very small difference in the Fe atom vibrations, reflecting a slightly smaller rigidity in the iron cores in human liver ferritin.

Common polymorphism (81Val>Ile) and rare mutations (257Arg>Ser and 335Ile>Ser) of the MC3R gene in obese Polish children and adolescents.

The predisposing role to human obesity of the MC3R gene polymorphism is controversial. In this report we present the first study focused on the search for the MC3R polymorphism in the Polish population. Altogether 257 obese children and adolescents (RBMI>120) and 94 adults, who were never obese or overweight (BMI<25), were studied. For all subjects the entire coding sequence was analyzed by direct DNA sequencing. One common polymorphism (81Val>Ile) and two rare mutations (257Arg>Ser and 335Ile>Ser) were identified. The common polymorphism was widely distributed in the obese and control cohorts, while the mutations were identified in four obese subjects only. In case of the 335Ile>Ser substitution a three-generation family, consisting of 20 members, was also analyzed. It was found that all carriers of the 335Ser mutation were obese, but among non-carriers obese subjects also were found. Our study suggests that the predisposing effect to obesity of the 81Ile polymorphic variant is rather unlikely. With regard to the studied rare mutations we suggest that the 335Ser allele may have a small predisposing effect.

Polymorphisms in the promoter region of the adiponectin (ADIPOQ) gene are presumably associated with transcription level and carcass traits in pigs.

The main goal of this study was to screen for polymorphisms in the porcine adiponectin (ADIPOQ) gene promoter, analyse their influence on transcription and identify any association with production traits in pigs. A 1018-bp region of the ADIPOQ gene promoter was analysed in 113 pigs, and seven novel polymorphisms found. Luciferase assays were performed in HEK293 (human embryonic kidney) cells and primary porcine adipose mesenchymal stem cells (pADMSCs) to investigate their affect on promoter activity. A 16-bp indel (c.-106_-91delGCCAGGGGTGTGAGCC) was found to influence promoter strength in vitro. In the HEK293 cell line, the Del/Del genotype showed greater luciferase activity than did the Ins/Ins genotype (P < 0.01). In pADMSCs, the insertion genotype of the ADIPOQ promoter showed greater luciferase activity than did the deletion genotype (P < 0.01). An association study performed for two novel polymorphisms, c.-67G>A and the 16-bp indel, showed significant correlation with loin measurements in Polish Landrace (P < 0.05) and synthetic line 990 (P < 0.01) pigs.

Three-dimensional arrangement of genes involved in lipid metabolism in nuclei of porcine adipocytes and fibroblasts in relation to their transcription level.

The 3-dimensional arrangement of chromosomes and genes within a nuclear space is considered to represent the level of transcriptional regulation. Understanding how the nuclear architecture of adipocyte cells contributes to gene expression has become the subject of great interest in the context of obesity research. In this study we investigated nuclear positioning of 3 gene loci involved in lipid metabolism in the pig (Sus scrofa, SSC) which is considered as an important animal model for obesity in humans. We found that the position of the SCD gene in the 3-dimensional space of the cell nucleus is not correlated with transcriptional activity. The gene locus as well as chromosome territory SSC14 occupied the same peripheral location in adipocyte and fibroblast cells, in spite of the fact that their transcription level differs significantly between both cell types. For the 2 other investigated genes, i.e. ACACA and SREBF1 and their chromosome territory (SSC12), slightly different nuclear locations were found. They occupied intermediate nuclear positions in fibroblast nuclei, while in adipocytes they were positioned in the nuclear interior. The more internal location of these genes corresponds to increased transcription levels in fat cells. Our results confirm the non-random position of genes and chromosome territories in nuclei of adult porcine cells and indicate that relationship between transcription activity and gene positioning exists only for some but not all genes.

Effect of three common SNPs in 5'-flanking region of LEP and ADIPOQ genes on their expression in Polish obese children and adolescents.

Genes encoding adipokines are considered as candidates for human obesity. In this study we analyzed the expression of leptin (LEP) and adiponectin (ADIPOQ) genes in relation to common 5'-flanking or 5'UTR variants: -2548G>A (LEP), 19A>G (LEP) and -11377C>G (ADIPOQ) in Polish obese children and adolescents. Relative transcription levels in the subcutaneous adipose tissue (real time RT-PCR) and serum protein concentrations (RIA) were measured in 48 obese subjects with known genotypes at three polymorphic sites and in five non-obese controls. None of the studied polymorphisms altered significantly the expression. Significantly elevated relative transcription levels of the LEP gene (P < 0.05) and serum leptin concentrations (P < 0.01) were recorded in obese patients, when compared with the non-obese controls, but such differences were not found for the ADIPOQ gene. Interestingly, the leptin to adiponectin protein concentration ratio (L/A) was approximately sevenfold higher in obese children and adolescents when compared with the non-obese controls (P < 0.001). Taking into consideration the observed relationship between the genotypes and the gene expression level we suggest that these SNPs are not conclusive markers for predisposition to obesity in Polish children and adolescents. On the other hand, we confirmed that the leptin to adiponectin gene expression ratio (L/A) is an informative index characterizing obesity.

Association of MC3R gene polymorphisms with body weight in the red fox and comparative gene organization in four canids.

There are five genes encoding melanocortin receptors. Among canids, the genes have mainly been studied in the dog (MC1R, MC2R and MC4R). The MC4R gene has also been analysed in the red fox. In this report, we present a study of chromosome localization, comparative sequence analysis and polymorphism of the MC3R gene in the dog, red fox, arctic fox and Chinese raccoon dog. The gene was localized by FISH to the following chromosome: 24q24-25 in the dog, 14p16 in the red fox, 18q13 in the arctic fox and NPP4p15 in the Chinese raccoon dog. A high identity level of the MC3R gene sequences was observed among the species, ranging from 96.0% (red fox--Chinese raccoon dog) to 99.5% (red fox--arctic fox). Altogether, eight polymorphic sites were found in the red fox, six in the Chinese raccoon dog and two in the dog, while the arctic fox appeared to be monomorphic. In addition, association of several polymorphisms with body weight was analysed in red foxes (the number of genotyped animals ranged from 319 to 379). Two polymorphisms in the red fox, i.e. a silent substitution c.957A>C and c.*185C>T in the 3'-flanking sequence, showed a significant association (P < 0.01) with body weight.

Vibrational properties of alpha- and sigma-phase Fe-Cr alloy.

Experimental and theoretical studies, of the Fe-partial phonon density of states (PDOS) for Fe52.5Cr47.5 alloy having alpha and sigma phases were carried out. The former using the nuclear resonant inelastic x-ray scattering method, and the latter with the direct one. Characteristic features of PDOS, which distinguish one phase from the other, were revealed and successfully reproduced by the theory. Data pertinent to the dynamics such as the Lamb-Mössbauer factor, f, the kinetic energy per atom, E(k), and the mean force constant, D, were directly derived, while vibrational specific heat at constant volume, C(V), and vibrational entropy, S were calculated using the Fe partial PDOS. Based on the values of f and C(V), we determined Debye temperatures, Theta(D). An excellent agreement for some quantities derived from experiment and first-principles theory, like C(V) and quite good ones for others like D and S were obtained.

Genetics of fat tissue accumulation in pigs: a comparative approach.

Fatness traits are important in pig production since they influence meat quality and fattening efficiency. On the other hand, excessive fat accumulation in humans has become a serious health problem due to worldwide spread of obesity. Since the pig is also considered as an animal model for numerous human diseases, including obesity and metabolic syndrome, comparative genomic studies may bring new insights into genetics of fatness/obesity. Input of genetic factors into phenotypic variability of these traits is rather high and the heritability coefficient (h(2)) of these traits oscillates around 0.5. Genome scanning revealed the presence of more than 500 QTLs for fatness in the pig genome. In addition to QTL studies, many candidate gene polymorphisms have been analyzed in terms of their associations with pig fatness, including genes encoding leptin (LEP) and its receptor (LEPR), insulin-like growth factor 2 (IGF-2), fatty acid-binding proteins (FABP3 and FABP4), melanocortin receptor type 4 (MC4R), and the FTO (fat mass and obesity-associated) gene. Among them, a confirmed effect on pig fatness was found for a well-known polymorphism of the IGF-2 gene. In humans the strongest association with predisposition to obesity was shown for polymorphism of the FTO gene, while in pigs such an association seems to be doubtful. The development of functional genomics has revealed a large number of genes whose expression is associated with fat accumulation and lipid metabolism, so far not studied extensively in terms of the association of their polymorphism with pig fatness. Recently, epigenomic mechanisms, mainly RNA interference, have been considered as a potential source of information on genetic input into the fat accumulation process. The rather limited progress in studies focused on the identification of gene polymorphism related with fatness traits shows that their genetic background is highly complex.

Unusual dynamics of Fe atoms in a chromium matrix.

(57)Fe site Mössbauer spectroscopy (MS) was used to investigate the dynamics of (57)Fe atoms embedded in a chromium lattice as impurities. From the Mössbauer spectra recorded in the temperature range of 80-350 K, a temperature dependence of the Lamb-Mössbauer factor, f, was determined. The latter revealed an unusual dynamics of (57)Fe atoms, namely a harmonic mode below T≈145 K with a characteristic effective Debye temperature Θ(eff) = 190.2 K and a strongly anharmonic one above T≈145 K. The latter mode exists in two clearly defined temperature intervals with significantly different Θ(eff) values, namely (i) ∼155 K for ∼145 K ≤ T ≤ ∼240 K and the record-high anharmonic coefficient ε = -25.8 × 10(-4) K(-1), and (ii) ∼151 K for T ≥ ∼240 K with ε = -14 × 10(-4) K(-1). Based on Visscher's theory, the record-low value of the relative binding force constant for Fe atoms was determined as 0.0997 for the harmonic regime. It is suggested that the unusual dynamics observed in this study might be related to the underlying spin-, charge- and strain-density waves of chromium.

Debye temperature of disordered bcc-Fe-Cr alloys.

The Debye temperature, Θ(D), of Fe(100-x)Cr(x) disordered alloys with 0 ≤ x ≤ 99.9 was determined from the temperature dependence of the centre shift of (57)Fe Mössbauer spectra recorded in the temperature range of 60-300 K. Its compositional dependence shows an interesting non-monotonous behaviour. For 0

The pig CART (cocaine- and amphetamine-regulated transcript) gene and association of its microsatellite polymorphism with production traits.

The cocaine- and amphetamine-regulated transcript (CART) gene is a candidate gene that may affect performance and body composition traits in the pig. The purpose of this study was to establish the chromosomal localization and genomic sequence of the porcine CART gene, search for polymorphism and analyse its phenotypic effect in 644 pigs representing two breeds, Polish Large White (PLW) and Polish Landrace (PL), and a synthetic line 990 (L990). The CART gene was fluorescence in situ hybridization (FISH)-mapped to the chromosome 16q21. The 1878 bp DNA fragment covering three exons, two introns and the 5' flanking region was sequenced and analysed. A new A/G single nucleotide polymorphism (SNP) at position -238 bp was found. The coding sequence was conserved between porcine and human CART genes. Previously unknown short tandem repeat polymorphism (CA)(2)(CG)(n)(CA)(n) was identified in intron 2. Three alleles 251, 253 and 259 bp were found. The 251-bp allele was predominant in all the analysed populations of pigs, whereas the 253-bp allele occurred with the lowest frequency. The statistical analysis revealed significant allelic additive effects on meat content in carcass (p < 0.05) and abdominal fat weight (p < 0.01) in PLW, and meat content in carcass (p < 0.05) and backfat thickness (p < 0.05) in PL. Our study confirmed that chromosome region harbouring the CART gene is a promising quantitative trait loci for pig production traits.

Electronic structure of a σ-FeCr compound.

The electronic structure of a σ-FeCr compound in a paramagnetic state was calculated for the first time in terms of isomer shifts and quadrupole splittings. The former were calculated using the charge self-consistent Korringa-Kohn-Rostoker (KKR) Green's function technique, while the latter were estimated from an extended point charge model. The calculated quantities combined with recently measured site occupancies were successfully used to analyze a Mössbauer spectrum recorded at room temperature using only five fitting parameters namely background, total intensity, linewidth, IS0 (necessary to adjust the refined spectrum to the used Mössbauer source) and the QS proportionality factor. Theoretically determined changes of the isomer shift for the σ-FeCr sample were found to be in line with the corresponding ones measured on a α-FeCr sample.

Preimplantation testing for chromosomal disorders improves reproductive outcome of poor-prognosis patients.

The clinical impact of PGD was evaluated through the analysis of the reproductive outcome before and after PGD in the same group of poor prognosis IVF patients, undergoing PGD for chromosomal abnormalities. Based on a series of 2359 PGD cycles, resulting in the establishment of 498 chromosomal abnormality-free clinical pregnancies, the reproductive history prior to PGD was analysed. Of 483 previous pregnancies analysed in patients with 432 pregnancies generated after PGD for aneuploidies, 328 (68%) ended in spontaneous abortions, in contrast to 28.4% after PGD, with only 155 (32%) resulting in deliveries, compared with 71.9% take-home baby rates after PGD. The patients experienced 315 previous IVF attempts, resulting in the transfer of 706 embryos in 308 cycles, of which only 49 (6.9%) implanted, compared with a 34.9% implantation rate observed in the same patients after PGD. Similar analysis of the previous reproductive outcomes of 45 carriers of balanced translocations achieving pregnancies following PGD, showed even stronger clinical impact, with a reduction of spontaneous abortions from 87.8% to 17.8%, and improvement of take-home baby rate from 11.5% to 81.4% after PGD. The results demonstrate a strong clinical impact of PGD, resulting in improvement of implantation rate, reduction of spontaneous abortions and increase in the take-home baby rate.

Frequency and distribution of chromosome abnormalities in human oocytes.

It was previously shown that more than half of the human oocytes obtained from IVF patients of advanced reproductive age are aneuploid, due to meiosis I and meiosis II errors. The present paper further confirms that 61.8% of the oocytes tested by fluorescent probes specific for chromosomes 13, 16, 18, 21 and 22 are abnormal, representing predominantly chromatid errors, which are the major source of aneuploidy in the resulting embryos. Almost half of the oocytes with meiosis I errors (49.3%) are prone to sequential meiosis II errors, which may lead to aneuploidy rescue in 30.8% of the cases. Half of the detected aneuploidies (49.8%) are of complex nature with involvement of two or more chromosomes, or the same chromosome in both meiotic divisions. The aneuploidy rates for individual chromosomes are different, with a higher prevalence of chromosome 21 and 22 errors. The origin of aneuploidy for the individual chromosomes is also not random, with chromosome 16 and 22 errors originating more frequently in meiosis II, and chromosome 18, 13 and 21 errors in meiosis I. There is an age dependence not only for the overall frequency of aneuploidies, but also for each chromosome error, aneuploidies originating from meiosis I, meiosis II, and both meiosis I and meiosis II errors, as well as for different types of aneuploidies. The data further suggest the practical relevance of oocyte aneuploidy testing for detection and avoidance from transfer of the embryos deriving from aneuploid oocytes, which should contribute significantly to the pregnancy outcomes of IVF patients of advanced reproduction age.

Conceptual "Heat-Driven" approach to the synthesis of DNA oligonucleotides on microarrays.

The discovery of deoxyribonucleoside cyclic N-acylphosphoramidites, a novel class of phosphoramidite monomers for solid-phase oligonucleotide synthesis, has led to the development of a number of phosphate protecting groups that can be cleaved from DNA oligonucleotides under thermolytic neutral conditions. These include the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, 3-(N-tert-butyl)carboxamido-1-propyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, and 4-methythiobutyl groups. When used for 5'-hydroxyl protection of nucleosides, the analogous 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group exhibited excellent thermolytic properties, which may permit an iterative "heat-driven" synthesis of DNA oligonucleotides on microarrays. In this regard, progress has been made toward the use of deoxyribonucleoside cyclic N-acylphosphoramidites in solid-phase oligonucleotide syntheses without nucleobase protection. Given that deoxyribonucleoside cyclic N-acylphosphoramidites produce oligonucleotides with heat-sensitive phosphate protecting groups, blocking the 5'-hydroxyl of these monomers with, for example, the thermolabile 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group may provide a convenient thermo-controlled method for the synthesis of oligonucleotides on microarrays.