An Improved Method and Device for Nucleic Acid Isolation Using a High-Salt Gel Electroelution Trap.

The success of DNA analytical methods, including long-read sequencing, depends on the availability of high-quality, purified DNA. Previously, we developed a method and device for isolating high-molecular-weight (HMW) DNA for long-read sequencing using a high-salt gel electroelution trap. Here, we present an improved version of this method for purifying nucleic acids with high yield and purity from even the most challenging biological samples. The proposed method is a significant improvement over the previously published procedure, offering a simple, fast, and efficient solution for isolating HMW DNA and smaller DNA and RNA molecules. The method utilizes vertical gel electrophoresis in two nested, partially overlapping electrophoretic columns. The upper, smaller-diameter column has a thin layer of agarose gel at the bottom, which separates nucleic acids from impurities, and an electrophoresis buffer on top. After the target nucleic acid has been gel-purified on the upper column, a larger-diameter column with a layer of high-salt gel overlaid with electrophoresis buffer is inserted from below. The purified nucleic acid is then electroeluted into the buffer-filled gap between the separating gel and the high-salt gel, where excess counterions from the high-salt gel slow its migration and cause it to accumulate. The proposed vertical purification system outperforms the previously described horizontal system in terms of ease of use, speed, scalability, and compatibility with high-throughput workflows. Furthermore, the vertical system allows for the sequential purification of several nucleic acid species from the same sample using interchangeable salt-gel columns.

Association of ADIPOQ Gene Polymorphisms with Type 2 Diabetes and Obesity Risk in the Kazakh Population: A Case-Control and Population-Based Study.

Type 2 diabetes mellitus (T2DM) is a socially significant disease with increasing prevalence worldwide. It is characterized by heterogeneous metabolic disorders and is associated with various risk factors, including BMI, abnormal lipid levels, hypertension, smoking, dietary preferences, physical inactivity, sedentary lifestyle, family history of diabetes, prediabetes or gestational diabetes, inflammation, intrauterine environment, age, sex, ethnicity, and socioeconomic status. Assessing the genetic risk of developing T2DM in specific populations remains relevant. The ADIPOQ gene, encoding adiponectin, is directly related to the risk of developing T2DM, obesity, and cardiovascular diseases. Our study demonstrated significant associations of ADIPOQ gene polymorphisms with the risk of developing T2DM and obesity, as well as with fasting glucose levels and BMI, in the Kazakh population. Specifically, rs266729 was significantly associated with T2DM and obesity in the Kazakh population, while other studied polymorphisms (rs1501299, rs2241766, and rs17846866) did not show a significant association. These findings suggest that ADIPOQ gene polymorphisms may influence T2DM risk factors and highlight the importance of genetic factors in T2DM development. However, further research in larger cohorts is needed to confirm these associations.

Transcriptome profiling and analysis of patients with esophageal squamous cell carcinoma from Kazakhstan.

Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer in Central Asia, often diagnosed at advanced stages. Understanding population-specific patterns of ESCC is crucial for tailored treatments. This study aimed to unravel ESCC's genetic basis in Kazakhstani patients and identify potential biomarkers for early diagnosis and targeted therapies. ESCC patients from Kazakhstan were studied. We analyzed histological subtypes and conducted in-depth transcriptome sequencing. Differential gene expression analysis was performed, and significantly dysregulated pathways were identified using KEGG pathway analysis (p-value < 0.05). Protein-protein interaction networks were constructed to elucidate key modules and their functions. Among Kazakhstani patients, ESCC with moderate dysplasia was the most prevalent subtype. We identified 42 significantly upregulated and two significantly downregulated KEGG pathways, highlighting molecular mechanisms driving ESCC pathogenesis. Immune-related pathways, such as viral protein interaction with cytokines, rheumatoid arthritis, and oxidative phosphorylation, were elevated, suggesting immune system involvement. Conversely, downregulated pathways were associated with extracellular matrix degradation, crucial in cancer invasion and metastasis. Protein-protein interaction network analysis revealed four distinct modules with specific functions, implicating pathways in esophageal cancer development. High-throughput transcriptome sequencing elucidated critical molecular pathways underlying esophageal carcinogenesis in Kazakhstani patients. Insights into dysregulated pathways offer potential for early diagnosis and precision treatment strategies for ESCC. Understanding population-specific patterns is essential for personalized approaches to ESCC management.

Molecular and seroepidemiological investigation of Сoxiella burnetii and spotted fever group rickettsiae in the southern region of Kazakhstan.

Ticks are involved in the circulation of a number of human pathogens, including spotted fever group (SFG) Rickettsia spp. and Coxiella burnetii. Little is known about the occurrence of these microorganisms in the southern region of Kazakhstan. In 2018-2022, a total of 726 ticks were collected from bitten humans, livestock, and vegetation in four oblasts of the southern region of Kazakhstan and subjected to DNA extraction. The overall infection rate of Coxiella spp. and Rickettsia spp. in the ticks was 3.3% (24/726) and 69.9% (300/429), respectively. Phylogenetic analysis of ompA and gltA genes revealed the presence of three pathogenic SFG rickettsiae: Candidatus R. tarasevichiae, R. aeschlimannii and R. raoultii in ticks collected from bitten humans. In addition, Candidatus R. barbariae was detected in six Rhipicephalus turanicus ticks for the first time in Kazakhstan. To determine the seroprevalence of C. burnetii infection, we performed a serological analysis of samples collected from 656 domestic ruminants (cattle, sheep, and goats) in the region. Overall, 23.5% (154/656) of the animals tested were positive for IgG against C. burnetii. Seroprevalence at the herd level was 54% (28/52). Goats (43%; 12/28; odds ratio (OD) = 28.9, p < 0.05) and sheep (31.9%; 137/430; OD = 18.1, p < 0.05) had higher seroprevalence than cattle (2.5%; 5/198). Among the risk factors considered in this study, age (p = 0.003) and the oblast in which the animals were sampled (p = 0.049) were statistically associated with seropostivity for Q fever in sheep, according to the results of multivariate logistic regression analysis. Seroprevalence ranged from 0% to 55.5% in animals in different districts of the southern region of Kazakhstan. Active C. burnetii bacteremia was detected in four of 154 (2.6%) seropositive animals. The data obtained provide strong evidence of the presence of pathogenic rickettsiae and C. burnetii in the southern region of Kazakhstan and emphasize the need to improve epidemiological surveillance in the region.

Theileria and Babesia infection in cattle - First molecular survey in Kazakhstan.

Central Asia, including Kazakhstan, is an endemic area of Theileria and Babesia infections in cattle. Current data on the geographic distribution, prevalence, and genetic diversity of these pathogens in vertebrate hosts are lacking in Kazakhstan. The present study aimed to fill this gap, using molecular techniques for the first time. A cross-sectional survey was performed on adult cattle from 40 villages in nine administrative districts of the provinces of Turkistan and Zhambyl, southern Kazakhstan, in summer 2020. A total of 766 blood samples were screened for Theileria annulata (enolase gene), Theileria orientalis (major piroplasm surface protein gene, MPSP) and Babesia spp. (18 S ribosomal RNA gene) using polymerase chain reaction. The genetic variability of Theileria spp. was assessed by sequencing one amplicon from each village. All Babesia spp. positive amplicons were sequenced to identify the species involved. The overall prevalence of infections with T. annulata, T. orientalis and Babesia spp. was 83.0% (40 villages positive), 33.3% (31 villages) and 13.5% (36 villages), respectively. Co-infections with two or three species were present in 48.9% of all positive cattle. Theileria annulata showing a high polymorphism of the enolase gene occurred with similar frequency in both provinces. Theileria orientalis was detected for the first time in Kazakhstan being significantly (P = 0.014) more prevalent in Zhambyl than in Turkistan. Fourteen genotypes of T. orientalis were identified; two belonged to the moderately virulent MPSP-type 1 ('Chitose') and the others to MPSP-type 3 ('Buffeli') which is considered avirulent. The prevalence of Babesia infection was significantly (P < 0.000) higher in Turkistan than in Zhambyl. An unequivocal identification of the species involved was possible in 127 sequenced samples: Babesia occultans was the most common species, followed by Babesia bigemina and Babesia major, the latter being the first record in the country. The results show that Theileria and Babesia infections in cattle are widespread and occur with remarkably high prevalence in the southern Kazakhstan. They also provide first data on the genetic diversity of the species involved.

Prevalence and genetic diversity of coronaviruses, astroviruses and paramyxoviruses in wild birds in southeastern Kazakhstan.

Wild birds are natural reservoirs of many emerging viruses, including some zoonoses. Considering that the territory of Kazakhstan is crossed by several bird migration routes, it is important to know pathogenic viruses circulating in migratory birds in this region. Therefore, the aim of this study was to identify the host range, diversity and spatial distribution of avian paramyxoviruses, coronaviruses, and astroviruses in free-ranging wild birds in the southeastern region of Kazakhstan. For this purpose, we collected tracheal and cloacal swabs from 242 wild birds belonging to 51 species and screened them using conventional PCR assays. Overall, 4.1% (10/242) and 2.9% (7/242) of all examined birds tested positive for coronaviruses and astroviruses, respectively. Coronaviruses were found in the orders Pelecaniformes (30%; 3/10), Charadriiformes (30%; 3/10), Columbiformes (20%; 2/10), Anseriformes (10%; 1/10), and Passeriformes (10%; 1/10). All detected strains belonged to the genus Gammacoronavirus. Astroviruses were detected in birds representing the orders Passeriformes (57%; 4/7), Coraciiformes (14%; 1/7), Charadriiformes (14%; 1/7), and Columbiformes (14%; 1/7). Paramyxoviruses were observed in only two birds (0.8%; 2/242). Both strains were closely related to the species APMV-22, which had not been previously detected in Kazakhstan. Phylogenetic analysis of the partial RdRp gene sequences of the virus strains revealed three different clades of astroviruses, two clades of coronaviruses, and one clade of paramyxoviruses. The results of this study provide valuable information on the diversity and spatial distribution of paramyxoviruses, coronaviruses, and astroviruses in wild birds in southeastern Kazakhstan and highlight the importance of further thorough monitoring of wild birds in this region.

Whole-Exome Sequencing Reveals Pathogenic SIRT1 Variant in Brain Arteriovenous Malformation: A Case Report.

Arteriovenous malformations of the brain (bAVMs) are plexuses of pathological arteries and veins that lack a normal capillary system between them. Intracranial hemorrhage (hemorrhagic stroke) is the most frequent clinical manifestation of AVM, leading to lethal outcomes that are especially high among children and young people. Recently, high-throughput genome sequencing methods have made a notable contribution to the research progress in this subject. In particular, whole-exome sequencing (WES) methods allow the identification of novel mutations. However, the genetic mechanism causing AVM is still unclear. Therefore, the aim of this study was to investigate the potential genetic mechanism underlying AVM. We analyzed the WES data of blood and tissue samples of a 30-year-old Central Asian male diagnosed with AVM. We identified 54 polymorphisms in 43 genes. After in-silica overrepresentation enrichment analysis of the polymorphisms, the SIRT1 gene variant (g.67884831C>T) indicated a possible molecular mechanism of bAVM. Further studies are required to evaluate the functional impact of SIRT1 g.67884831C>T, which may warrant further replication and biological investigations related to sporadic bAVM.

Activated leukocyte cell adhesion molecule/cluster of differentiation 166 rs10933819 (G>A) variant is associated with familial intracranial aneurysms.

Rupture of intracranial aneurysms (IAs) is the most common cause of subarachnoid hemorrhage (SAH). Currently, there is sufficient evidence to indicate that inflammatory responses contribute to aneurysm rupture. Moreover, the familial occurrence of SAH suggests that genetic factors may be involved in disease susceptibility. In the present study, a clinically proven case of IA in a patient who is a heterozygous mutation carrier of the activated leukocyte cell adhesion molecule (ALCAM)/cluster of differentiation 166 (CD166) gene, is reported. Genomic DNA was extracted from two siblings diagnosed with SAH and other available family members. A variant prioritization strategy that focused on functional prediction, frequency, predicted pathogenicity, and segregation within the family was employed. Sanger sequencing was also performed on the unaffected relatives to assess the segregation of variants within the phenotype. The verified mutations were sequenced in 145 ethnicity-matched healthy individuals. Based on whole exome sequencing data obtained from three individuals, two of whom were diagnosed with IAs, the single-nucleotide variant rs10933819 was prioritized in the family. Only one variant, rs10933819 (G>A), in ALCAM co-segregated with the phenotype, and this mutation was absent in ethnicity-matched healthy individuals. Collectively, ALCAM c1382 G>A p.Gly229Val was identified, for the first time, as a pathogenic mutation in this IA pedigree.

Designing Allele-Specific Competitive-Extension PCR-Based Assays for High-Throughput Genotyping and Gene Characterization.

Polymerase chain reaction (PCR) is a simple and rapid method that can detect nucleotide polymorphisms and sequence variation in basic research applications, agriculture, and medicine. Variants of PCR, collectively known as allele-specific PCR (AS-PCR), use a competitive reaction in the presence of allele-specific primers to preferentially amplify only certain alleles. This method, originally named by its developers as Kompetitive Allele Specific PCR (KASP), is an AS-PCR variant adapted for fluorescence-based detection of amplification results. We developed a bioinformatic tool for designing probe sequences for PCR-based genotyping assays. Probe sequences are designed in both directions, and both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) may be targeted. In addition, the tool allows discrimination of up to four-allelic variants at a single SNP site. To increase both the reaction specificity and the discriminative power of SNP genotyping, each allele-specific primer is designed such that the penultimate base before the primer's 3' end base is positioned at the SNP site. The tool allows design of custom FRET cassette reporter systems for fluorescence-based assays. FastPCR is a user-friendly and powerful Java-based software that is freely available (http://primerdigital.com/tools/). Using the FastPCR environment and the tool for designing AS-PCR provides unparalleled flexibility for developing genotyping assays and specific and sensitive diagnostic PCR-based tests, which translates into a greater likelihood of research success.

Detection of Recombinant Proteins SOX2 and OCT4 Interacting in HEK293T Cells Using Real-Time Quantitative PCR.

In vivo biotinylation using wild-type and mutants of biotin ligases is now widely applied for the study of cellular proteomes. The commercial availability of kits for the highly efficient purification of biotinylated proteins and their excellent compatibility with LC-MS/MS protocols are the main reasons for the choice of biotin ligases. Since they are all enzymes, however, just a very low expression in cells is required for experiments. Therefore, it can be difficult to perform the quantifications of these enzymes in various samples. Traditional methods, such as western blotting, are not always fit for the detection of the expression levels. Therefore, real-time qRT-PCR, a technology that is more sensitive, was used in this study to quantify the expression of BirA fusions. Using this method, we detected high expression levels of BirA fusions in models of interactions of pluripotency transcription factors to carry out their relative quantification. We also found the absence of the competing endogenous proteins SOX2 and OCT4, as well as no cross-reactivity between BAP/BirA and the endogenous biotinylation system in HEK293T cells. Thus, these data indicated that the high level of biotinylation is due to the in vivo interaction of BAP-X and BirA-Y (X,Y = SOX2, OCT4) in the cell rather than their random collision, a big difference in the expression level of BirA fusions across samples or endogenous biotinylation.

Mitochondrial DNA analysis of ancient sheep from Kazakhstan: evidence for early sheep introduction.

Kazakhstan covers a vast territory, and it has always been a land of nomadic pastoralism, where domesticated horses and sheep were moved by nomadic people across the steppe. Previous reports suggest that sheep breeds from Kazakhstan have an intermediate genetic composition between Asian and European breeds; however, this data appears to be limited. Therefore, we studied the genetic diversity of ancient domestic sheep from two Late Bronze Age settlements, Toksanbai and Kent, located in the Pre-Caspian region of Kazakhstan and central Kazakhstan, respectively. We have applied ZooMS analysis for taxonomic identification of small ruminant remains to select ancient specimens of domestic sheep (Ovis aries). To assign sheep mitochondrial DNA (mtDNA) haplogroups, the single nucleotide polymorphisms (SNPs) from the control region were analyzed by real-time PCR and direct sequencing. Identical distribution of mtDNA haplogroups A (8/14; 57%), B (5/14; 36%), and C (1/14; 7%) was observed in the specimens from Toksanbai (n = 14) and Kent (n = 14). Ovine haplogroup A was predominant in both settlements. Both archeological sites had similar patterns of haplogroup distribution, indicating early sheep introduction into the region. These results are important to gain a better understanding of sheep migrations in the Eurasian steppe and highlight the importance of genomic analysis of earlier local lineages.