Point field emission electron source with a magnetically focused electron beam.

This paper presents a field emitter in the form of a silicon tip covered with a layer of carbon nanotubes. The emitted beam is focused with a set of two electrostatic lenses and - which is novelty in such structures - with a magnetic field. The presented approach gave very promising results. The field emitter was able to provide a high emission current (about 50 µA) and a beam with a small and homogeneous spot. Such electron sources are necessary components of many miniature MEMS and nanoelectronics devices. The presented source is dedicated especially for the use in currently developed MEMS X-ray sources and MEMS electron microscopes.

Molecular Characterisation of Uterine Endometrial Proteins during Early Stages of Pregnancy in Pigs by MALDI TOF/TOF.

The molecular mechanism underlying embryonic implantation is vital to understand the correct communications between endometrium and developing conceptus during early stages of pregnancy. This study's objective was to determine molecular changes in the uterine endometrial proteome during the preimplantation and peri-implantation between 9 days (9D), 12 days (12D), and 16 days (16D) of pregnant Polish Large White (PLW) gilts. 2DE-MALDI-TOF/TOF and ClueGOTM approaches were employed to analyse the biological networks and molecular changes in porcine endometrial proteome during maternal recognition of pregnancy. A total of sixteen differentially expressed proteins (DEPs) were identified using 2-DE gels and MALDI-TOF/TOF mass spectrometry. Comparison between 9D and 12D of pregnancy identified APOA1, CAPZB, LDHB, CCT5, ANXA4, CFB, TTR upregulated DEPs, and ANXA5, SMS downregulated DEPs. Comparison between 9D and 16D of pregnancy identified HP, APOA1, ACTB, CCT5, ANXA4, CFB upregulated DEPs and ANXA5, SMS, LDHB, ACTR3, HP, ENO3, OAT downregulated DEPs. However, a comparison between 12D and 16D of pregnancy identified HP, ACTB upregulated DEPs, and CRYM, ANXA4, ANXA5, CAPZB, LDHB, ACTR3, CCT5, ENO3, OAT, TTR down-regulated DEPs. Outcomes of this study revealed key proteins and their interactions with metabolic pathways involved in the recognition and establishment of early pregnancy in PLW gilts.

Effects of Three-Month Administration of High-Saturated Fat Diet and High-Polyunsaturated Fat Diets with Different Linoleic Acid (LA, C18:2n-6) to α-Linolenic Acid (ALA, C18:3n-3) Ratio on the Mouse Liver Proteome.

The aim of the study was to evaluate the effect of different types of high-fat diets (HFDs) on the proteomic profile of mouse liver. The analysis included four dietary groups of mice fed a standard diet (STD group), a high-fat diet rich in SFAs (SFA group), and high-fat diets dominated by PUFAs with linoleic acid (LA, C18:2n-6) to α-linolenic acid (ALA, C18:3n-3) ratios of 14:1 (14:1 group) and 5:1 (5:1 group). After three months of diets, liver proteins were resolved by two-dimensional gel electrophoresis (2DE) using 17 cm non-linear 3-10 pH gradient strips. Protein spots with different expression were identified by MALDI-TOF/TOF. The expression of 13 liver proteins was changed in the SFA group compared to the STD group (↓: ALB, APOA1, IVD, MAT1A, OAT and PHB; ↑: ALDH1L1, UniProtKB-Q91V76, GALK1, GPD1, HMGCS2, KHK and TKFC). Eleven proteins with altered expression were recorded in the 14:1 group compared to the SFA group (↓: ARG1, FTL1, GPD1, HGD, HMGCS2 and MAT1A; ↑: APOA1, CA3, GLO1, HDHD3 and IVD). The expression of 11 proteins was altered in the 5:1 group compared to the SFA group (↓: ATP5F1B, FTL1, GALK1, HGD, HSPA9, HSPD1, PC and TKFC; ↑: ACAT2, CA3 and GSTP1). High-PUFA diets significantly affected the expression of proteins involved in, e.g., carbohydrate metabolism, and had varying effects on plasma total cholesterol and glucose levels. The outcomes of this study revealed crucial liver proteins affected by different high-fat diets.

Identification of Differentially Expressed Gene Transcripts in Porcine Endometrium during Early Stages of Pregnancy.

During the early stages of pregnancy, the uterine endometrium undergoes dramatic morphologic and functional changes accompanied with dynamic variation in gene expression. Pregnancy-stage specific differentially expressed gene (DEG)-transcript-probes were investigated and identified by comparing endometrium transcriptome at 9th day (9D), 12th day (12D) and 16th day (16D) of early pregnancy in Polish large-white (PLW) gilts. Endometrium comparisons between 9D-vs-12D, 9D-vs-16D and 12D-vs-16D of early pregnancy identified 6049, 374 and 6034 highly significant DEG-transcript-probes (p < 0.001; >2 FC). GO term enrichment analysis identified commonly shared upregulated endometrial DEG-transcript-probes (p < 0.001; >2 FC), that were regulating the gene functions of anatomic structure development and transport (TG), DNA-binding and methyltransferase activity (ZBTB2), ion-binding and kinase activity (CKM), cell proliferation and apoptosis activity (IL1B). Downregulated DEG-transcript-probes (p < 0.001; >2 FC) were involved in regulating the gene functions of phosphatase activity (PTPN11), TC616413 gene-transcript and Sus-scrofa LOC100525539. Moreover, blastn comparison of microarray-probes sequences against sus-scrofa11 assembly identified commonly shared upregulated endometrial DEG-transcript-probes (E < 0.06; >2 FC), that were regulating the gene functions of reproduction and growth (SELENOP), cytoskeleton organization and kinase activity (CDC42BPA), phosphatase activity (MINPP1), enzyme-binding and cell-population proliferation (VAV3), cancer-susceptibility candidate gene (CASC4), cytoskeletal protein-binding (COBLL1), ion-binding, enzyme regulator activity (ACAP2) Downregulated endometrial DEG-transcript-probes (E < 0.06; >2FC) were involved in regulating the gene functions of signal-transduction (TMEM33), catabolic and metabolic processes (KLHL15). Microarray validation experiment on selected candidate genes showed complementarity to significant endometrial DEG-transcript-probes responsible for the regulation of immune response (IL1B, S100A11), lipid metabolism (FABP3, PPARG), cell-adhesion (ITGAV), angiogenesis (IL1B), intercellular transmission (NMB), cell-adhesion (OPN) and response to stimuli (RBP4) was confirmed by RT-PCR. This study provides a clue that identified pregnancy-stage specific microarray transcript probes could be considered as candidate genes for recognition and establishment of early pregnancy in the pig.

Cytogenetic and genetic study of a Y-linked microsatellite polymorphism in Polish Black-and-White cattle breed.

The aim of the current study was to characterize Polish Black-and-White cattle by morphological study of the Y chromosome. A total of 14 Y-linked microsatellites from UMN and INRA group were genotyped and assessed for polymorphism in a total 22 bulls. Cytogenetic studies in Polish Black-and-White bulls showed the existence of two morphological forms of Y chromosome. Among the 22 karyotypic analyzed bulls, 12 had submetacentric and 10 metacentric Y chromosome. The centromeric index of Y chromosome measured as percentage length of the p arm to total length ratio in the first case was 28 ± 3.97% and in the second 47 ± 7.28%, whereas the relative size of these chromosomes remained within the same range. Morphology and G- and C-banding patterns of both forms of Y chromosome were typical for other cattle breeds originating from Bos taurus. Out of a total of 14 microsatellite loci examined, 13 showed specific alleles for two forms of Y chromosome. In a pool of 62 alleles, 43 (69.3%) were common in the two groups of cattle, 19 (30.7%) can be considered as specific for the group; among them 8 were typical for metacentric group of Y chromosome and 11 for submetacentric.

A porcine gluteus medius muscle genome-wide transcriptome analysis: dietary effects of omega-6 and omega-3 fatty acids on biological mechanisms.

BACKGROUND: The level of omega-6 and omega-3 polyunsaturated fatty acids can affect many cellular systems and function via nuclear receptors or the bioactive lipid regulation of gene expression. The objective of this study was to investigate changes in the muscle transcriptome and the biological functions regulated by increased consumption of omega-3 and omega-6 fatty acids in the pig gluteus medius muscle. RESULTS: The transcriptome of the gluteus medius muscle was studied for pigs subjected to either a control diet or a diet supplemented with linseed and rapeseed oil to increase polyunsaturated fatty acid content. Next-generation sequencing (NGS) was used to generate the muscle tissue transcriptome database pointing differentially expressed genes (DEG). Comparative expression analyses identified 749 genes significantly differing at least in the twofold of change between two groups of animals fed with divergent level of omega-3 and omega-6 fatty acids. The expression of 219 genes was upregulated, and the expression of 530 genes was downregulated in the group of pigs supplemented with omega-3 and omega-6 fatty acids in relation to control group pigs. Results of RNA-seq indicated a role of fatty acid in the regulation of the expression of genes which are essential for muscle tissue development and functioning. Functional analysis revealed that the identified genes were important for a number of biological processes including inflammatory response, signaling, lipid metabolism, and homeostasis. CONCLUSIONS: Summarizing, obtained results provide strong evidence that omega-6 and omega-3 fatty acids regulate fundamental metabolic processes in muscle tissue development and functioning.

Effect of a diet enriched with omega-6 and omega-3 fatty acids on the pig liver transcriptome.

The optimal ratio of omega-6 to omega-3 polyunsaturated fatty acids (PUFAs) is important for keeping the homeostasis of biological processes and metabolism, yet the underlying biological mechanism is poorly understood. The objective of this study was to identify changes in the pig liver transcriptome induced by a diet enriched with omega-6 and omega-3 fatty acids and to characterize the biological mechanisms related to PUFA metabolism. Polish Landrace pigs (n = 12) were fed diet enriched with linoleic acid (LA, omega-6) and α-linolenic acid (ALA, omega-3) or standard diet as a control. The fatty acid profiling was assayed in order to verify how feeding influenced the fatty acid content in the liver, and subsequently next-generation sequencing (NGS) was used to identify differentially expressed genes (DEG) between transcriptomes between dietary groups. The biological mechanisms and pathway interaction networks were identified using DAVID and Cytoscape tools. Fatty acid profile analysis indicated a higher contribution of PUFAs in the liver for LA- and ALA-enriched diet group, particularly for the omega-3 fatty acid family, but not omega-6. Next-generation sequencing identified 3565 DEG, 1484 of which were induced and 2081 were suppressed by PUFA supplementation. A low ratio of omega-6/omega-3 fatty acids resulted in the modulation of fatty acid metabolism pathways and over-representation of genes involved in energy metabolism, signal transduction, and immune response pathways. In conclusion, a diet enriched with omega-6 and omega-3 fatty acids altered the transcriptomic profile of the pig liver and would influence animal health status.

Study of the differential transcription in liver of growth hormone receptor (GHR), insulin-like growth factors (IGF1, IGF2) and insulin-like growth factor receptor (IGF1R) genes at different postnatal developmental ages in pig breeds.

The objective of this study was to determine hepatic expression levels of GHR, IGF1R, IGF1 and IGF2 genes in young growing gilts at different developmental ages (60-210 days) in five pig breeds: Polish Large White (PLW), Polish Landrace (PL), Pulawska (Pul), Duroc (Dur) and Pietrain (Pie). We studied the differences among pig breeds as well as within each breed for pigs in different developmental ages. Obtained results revealed major differences among breeds in hepatic gene expression of porcine GHR, IGF1R, IGF1 and IGF2 genes in different developmental ages. The differences among breeds of GHR expression were significantly higher in PLW, PL at the age of 60, 90, 120 days as compared to Pul, Dur and Pie. In turn, the highest level of IGF1R expression was observed in PL at age of 150, 180 and 210 days, whereas in case of IGF1 the highest level was recorded in Pie gilts at the age of 60 and 90 days. Moreover trait associated study revealed highly significant correlations between hepatic expressions of IGF1R and IGF2 genes and carcass composition traits (P < 0.01) The results of study suggest that porcine GHR, IGF1R, IGF1 and IGF2 genes may be potential candidate genes for postnatal growth and carcass composition traits. Therefore, the implementation of the hepatic expression of GH/IGF genes into the pig breeding and gene assisted selection program in different pig breeds should be considered. However, further population wide study is needed to clarify the hepatic expression association with economic traits, such as body growth, meat quality and carcass composition traits.

The study of polymorphism within the promoter region of the osteopontin (OPN) gene in sows.

OBJECTIVES: The aim of the present study was to find a possible polymorphism in the promoter region of the osteopontin (OPN) gene, as a potential mutation region, connected with the transcription factor-binding sites or regulatory sequences and to estimate the expression of this gene in ovaries and oviduct of sows. MATERIAL AND METHODS: Sixty wbp x pbz sows after the first mating were slaughtered and tissues samples of the ovaries and oviduct were taken. Primer pairs for PCR analysis were designed on the basis of swine osteopontin 5' end sequence driven from GenBank. This study's amplified DNA fragment, spanning 274 bp of the promoter region, was chosen because of its contents of tree specific sites: type II collagen silence sequence (CACCTCC) at -682 (from the transcription initiation site), glucocortIcoid response site (TGTCCT) at -658 and CAAT box -592. This DNA fragment was subjected to a MSSCP analysis. RESULTS: A different MSSCP pattern was shown which indicates that mutation is located in this region. The samples of different conformers were sequenced and the A --> G transitions was identified in two positions -617 and -608. Restriction analysis of the DNA was performed, but unfortunately none of the known restriction enzymes recognized the novel SNPs, which is why the specific primer pairs characteristic for nucleotide A or for nucleotide G were chosen. In the second stage of the presented study the total RNA was extracted from the tissues of the ovaries and oviduct and the complementary DNA (cDNA) was synthesized. CONCLUSION: The real-time PCR analysis to determined the expression dynamics of the OPN gene in pig tissues was performed.

A new SNP in the promoter region of the porcine MYF5 gene has no effect on its transcript level in m. longissimus dorsi.

Myogenic factor 5 (myf-5) is the product of the MYF5 gene, belonging to the MyoD family. This transcription factor participates in the control of myogenesis. We identified 3 new mutations in the promoter region of the gene: A65C, C580T and C613T. The aim of this study was to evaluate the influence of the A65C transversion on gene expression. The analysis was conducted on 15 Polish Large White gilts. The relative content of MYF5 mRNA in m. longissimus dorsi did not differ significantly across MYF5 genotypes (AA, AC, CC). This result suggests that the A65C transversion may not play an important role in the expression of the MYF5 gene in analysed adult muscle but it abolishes a putative binding site for two transcription factors (CDP and HSF1) and creates such a site for Sp1.

New SNPs in the coding and 5' flanking regions of porcine MYOD1 (MYF3) and MYF5 genes.

Myogenic factor 3 (myf - 3) and myogenic factor 5 (myf - 5) are the products of genes: MYOD1 (MYF3) and MYF5, respectively, which belong to the MyoD family. These transcription factors control the processes of myogenesis. The fragments of both the genes comprising exons and promoters were amplified and sequenced. In the 5'UTR region of gene MYOD1, the G302A transition was identified and it is not recognized by any restriction endonuclease. In the promoter region of gene MYF5 we identified three mutations at positions: A65C (PCR-RFLP/AciI); C580T (PCR-RFLP/FokI) and C613T (PCR-RFLP/HinPI). Mutations C580T and C613T were characteristic for Pietrain x (Polish Large White x Polish Landrace) crossbred pigs named Torhyb. The C2931T transition, which is not recognized by any restriction enzyme, was identified in exon 3 of gene MYF5. This mutation results in a change of the amino acid sequence (Leu-->Pro). The frequency of particular genotypes at the MYOD1 and MYF5 loci proved to be dependent on pig breed. However, Duroc pigs were monomorphic at all the SNPs presented in this study. These SNPs might be analyzed in a further study as probably influencing carcass meatiness.