A genome-wide association study identifies novel candidate genes for susceptibility to diabetes mellitus in non-obese cats

Diabetes mellitus (DM) is a common feline endocrinopathy, which is similar to human type 2 diabetes (T2DM) in terms of its pathophysiology. T2DM occurs due to peripheral insulin resistance and/or β-cell dysfunction. Several studies have identified genetic and environmental factors that contribute to susceptibility to human T2DM. In cats, environmental factors such as obesity and physical inactivity have been linked with DM, although to date, the only genetic association that has been demonstrated is with a polymorphism in the feline MC4R gene. The aim of this study was to perform a genome-wide association study (GWAS) to identify polymorphisms associated with feline DM. Illumina Infinium 63k iSelect DNA arrays were used to analyse genomic DNA samples from 200 diabetic domestic shorthair cats and 399 non-diabetic control cats. Data was analysed using PLINK whole genome data analysis toolset. A linear model analysis, EMMAX, was done to test for population structure and HAPLOVIEW was used to identify haplotype blocks surrounding the significant SNPs to assist with candidate gene nomination. A total of 47,497 SNPs were available for analysis. Four SNPs were identified with genome-wide significance: chrA2.4150731 (praw = 9.94 x10-8); chrUn17.115508 (praw = 6.51 x10-8); chrUn17.394136 (praw = 2.53 x10-8); chrUn17.314128 (praw = 2.53 x10-8) as being associated with DM. The first SNP is located within chromosome A2, less than 4kb upstream of the dipeptidyl-peptidase-9 (DPP9) gene, a peptidase involved in incretin inactivation. The remaining three SNPs are located within a haplotype block towards the end of chromosome A3; within this region, genes of interest include TMEM18 and ACP1, both previously associated with T2DM. This study indicates a polygenic component to susceptibility to DM in cats and has highlighted several loci and candidate genes worthy of further investigation.

PSXV-10 Study of allelic pattern of PLAG1 gene in the historical and modern populations of two oldest Russian dairy cattle breeds

Increasing animal stature is one of the goals of modern breeding programs for many dairy cattle breeds, because stature related to higher milk yield. The PLAG1 gene was shown to be a strong candidate responsible for stature in different cattle breeds. The polymorphic SNP BovineHD1400007259, located within PLAG1 gene, is considered as a causal mutation responsible for stature. The aim of our work was to evaluate the effect of long selection for the increased body height on the alterations of the allele’s frequencies of the PLAG1 gene in the historical and modern populations of the Russian Yaroslavl and Kholmogor dairy cattle breeds. The historical specimens of Yaroslavl (n = 22) and Kholmogor (n = 12) cattle dated by the first quarter of the 20th century were derived from the craniological collection of the E.F. Liskun Museum for Animal Husbandry. The modern representatives of Yaroslavl (n = 31) and Kholmogor (n = 25) breeds were used for comparison. All works with historical samples were performed in dedicated facility of the L.K. Ernst Research Center for Animal Husbandry. The samples were genotyped using high-density DNA arrays (Illumina Inc., USA). The historical DNA was treated by USER enzyme before genotyping to avoid the misincoporated nucleotides occurred due to postmortem DNA damage. We observed significant differences in allele frequencies of PLAG1 genes between historical and modern populations of both breeds. The frequencies of G allele, which is associated with higher stature, were increased from 0.114 in historical Yaroslavl cattle and from 0.167 in historical Kholmogor cattle to 0.633 and 0.860 in the modern breeds’ representatives, respectively. Our data suggest that PLAG1 gene was affected by artificial selection in studied cattle breeds. The research results will be useful for elucidation of the history of these two oldest Russian dairy cattle breeds. The study was funded by the RSF No. 21-66-00007.

PSXII-21 Genome-wide search for genomic regions under putative selection in two Russian native cattle breeds using high-density SNP Bead Chip

National livestock genetic resources are the valuable source of genetic variability, which are necessary to ensure the sustainability of the local animal production systems. The aim of our work was to characterize the genomic architecture and to detect signatures of selection in two oldest Russian native cattle breeds, using high-density DNA arrays. Fifty-six animals of Yaroslavl (n = 31) and Kholmogor (n = 25) were selected for the study. The Illumina Bovine HD BeadChip (777,962 SNPs) was used for genotyping. High-density SNP genotypes for Holsteins (n = 25) were used as the reference. Three methods (Fst, hapFLK, and ROH) were implemented to detect the genomic regions to be under putative selection. The results of the PCA-plot, Neighbor-Net analysis and Admixture clustering showed clear genetic differences between Yaroslavl and Kholmogor breeds as well as between them and Holsteins. Comparison of the results produced by different methods showed presence of twelve regions in genome of Yaroslavl breed and nine regions in genome of Kholmogor breeds, which were identified at least by two methods. We have confirmed nine regions under putative selection in genome of Yaroslavl cattle and six regions in genome of Kholmogor cattle, which were described previously based on medium-density SNP genotypes, herewith the flanking positions of the most of these regions were expanded. Additionally, we detected three new putative genomic regions affected by selection in each studied breed, which were localized on chromosomes 4, 15 and 17 in Yaroslavl breed and on chromosomes 12, 15 and 18 in Kholmogor breed. The functional annotation of genes localized within identified regions was carried out. Our research results can be useful for genetic improvement of the studied cattle breeds and for developing the programs for their conservation. The study was supported by RSF within project No. 19-76-20012.

PSX-17 Genome-wide diversity and demographic history of Russian native goat breeds

Specific environmental conditions and local livestock management systems resulted in creation of valuable native breeds. The timely monitoring of genetic diversity within native breeds based on using high-throughput DNA arrays will prevent their irreparable loss. In this regard, we aimed to assess genome-wide diversity and to study demographic history of Russian native goat breeds (Altai Mountain, Orenburg, Soviet Mohair, Dagestan Milk, Dagestan Local, Dagestan Fluff and Karachaev) based on SNP-data. A total of 200 goats were genotyped using Goat 50K SNP BeadChip (Illumina, USA). Quality control and SNP-filtering were performed in PLINKv1.9. R package ‘diveRsity’ was used to calculate observed heterozygosity (Ho), expected heterozygosity (He), and inbreeding coefficient (Fis). Effective population sizes (Ne) were estimated in SneP software. Observed heterozygosity was high and exceeded 0.402 in five out of seven breeds. Orenburg, Soviet Mohair, Dagestan Milk, and Karachaev breeds showed slight excess of heterozygotes varied from 0.6% (Fis= -0.015) in Orenburg to 1.7% (Fis= -0.04) in Karachaev breed. The traces of insignificant inbreeding were found in Dagestan Local (Fis=0.005) and Dagestan Fluff (Fis= 0.01) breeds. The recent effective population sizes estimated for four generations ago varied from 140 in Karachaev to 472 in Orenburg breed. Analysis of historical trends in effective population sizes estimated for sixty generations ago revealed obvious decrease ranging from 10.25% in Dagestan Local to 34.65% in Orenburg breed. However, recent effective sizes in Russian native goats are higher than critical threshold (Ne= 100) that is essential to breed maintenance in the future. Our research findings provide an evidence that Russian native goat breeds are not in endangered status, but development of the effective utilization programs is highly recommended. The genotyping of 96 goats was funded by RSF No. 19-76-20006. The reported study was funded by RFBR according to the research project № 18-316-20006.

Graphene Templated DNA Arrays and Biotin-Streptavidin Sensitive Bio-Transistors Patterned by Dynamic Self-Assembly of Polymeric Films Confined within a Roll-on-Plate Geometry

Patterning of surfaces with a simple strategy provides insights into the functional interfaces by suitable modification of the surface by novel techniques. Especially, highly ordered structural topographies and chemical features from the wide range of interfaces have been considered as important characteristics to understand the complex relationship between the surface chemistries and biological systems. Here, we report a simple fabrication method to create patterned surfaces over large areas using evaporative self-assembly that is designed to produce a sacrificial template and lithographic etch masks of polymeric stripe patterns, ranging from micrometer to nanoscale. By facilitating a roll-on-plate geometry, the periodically patterned surface structures formed by repetitive slip-stick motions were thoroughly examined to be used for the deposition of the Au nanoparticles decorated graphene oxide (i.e., AuNPs, ~21 nm) and the formation of conductive graphene channels. The fluorescently labeled thiol-modified DNA was applied on the patterned arrays of graphene oxide (GO)/AuNPs, and biotin-streptavidin sensitive devices built with graphene-based transistors (GFETs, effective mobility of ~320 cm2 V−1 s−1) were demonstrated as examples of the platform for the next-generation biosensors with the high sensing response up to ~1 nM of target analyte (i.e., streptavidin). Our strategy suggests that the stripe patterned arrays of polymer films as sacrificial templates can be a simple route to creating highly sensitive biointerfaces and highlighting the development of new chemically patterned surfaces composed of graphene-based nanomaterials.

Advantageous Antibody Microarray Fabrication Through DNA-Directed Immobilization: A Step Toward Use of Extracellular Vesicles in Diagnostics

An optimized general protocol for DNA-protein ligation is provided and the conjugates are used to convert DNA arrays into antibody microarrays. Arrays obtained through DDI were used to capture and characterize extracellular vesicles (EVs), an emerging class of biomarkers. The proposed platform was tested against commercially available antibody microarrays, showing good performance combined with ease of fabrication.

Advantageous Antibody Microarray Fabrication Through DNA-Directed Immobilization: A Step Toward Use of Extracellular Vesicles in Diagnostics

An optimized general protocol for DNA-protein ligation is provided and the conjugates are used to convert DNA arrays into antibody microarrays. Arrays obtained through DDI were used to capture and characterize extracellular vesicles (EVs), an emerging class of biomarkers. The proposed platform was tested against commercially available antibody microarrays, showing good performance combined with ease of fabrication.

RbAp46/48LIN-53 and HAT-1 are required for initial CENP-AHCP-3 deposition and de novo centromere formation in Caenorhabditis elegans embryos

ABSTRACTForeign DNA microinjected into the Caenorhabditis elegans germline forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Injected linear, short DNA fragments concatemerize into high molecular weight (HMW)-DNA arrays that are visible as punctate DAPI-stained foci in oocytes, which undergo chromatinization and centromerization in embryos. The inner centromere, inner and outer kinetochore components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, assemble onto the nascent ACs during the first mitosis. Yet, due to incomplete DNA replication of the nascent ACs, centromeric proteins are not oriented at the poleward faces of the nascent ACs in mitosis, resulting in lagging ACs. The DNA replication efficiency of ACs improves over several cell cycles. We found that a condensin subunit, SMC-4, but not the replicative helicase component, MCM-2, facilitates de novo CENP-AHCP-3 deposition on nascent ACs. Furthermore, H3K9ac, H4K5ac, and H4K12ac are highly enriched on newly chromatinized ACs. HAT-1 and RbAp46/48LIN-53, which are essential for de novo centromere formation and segregation competency of nascent ACs, also hyperacetylate histone H3 and H4. Different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53, RbAp46/48LIN-53 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs.

Experimental Evidence Supports New Perspectives in Homeopathy

The contentious debate between homeopathy and orthodox medicine has been due to the fact that homeopathy is founded on a heuristic philosophy that is not justified by contemporary scientific evidence. In this context, however, two pillars of the method, that is, serial dilution and succussion, are poorly understood in orthodox pharmacology. The experimental data collected in the last 10 years, by means of electronic microscopy, electron diffraction and DNA arrays investigations, are consistent with the presence of nanoparticles (nanoassociates) in homeopathic medicines and seem to provide a coherent view of the essence of the homeopathy discipline, superseding all previous speculative interpretations. An acceptance of this new evidence is here suggested to remove, in principle, the barrier that separates the conventional and homeopathic therapeutic methods, and to offer new and important perspectives on future health care.