Bacterial poly(A) polymerases (PAPs) play an important role in RNA metabolism but remain poorly characterized outside Gammaproteobacteria. Here, we cloned and biochemically characterized the first PAP from Alphaproteobacteria, specifically from Marinobacter lipolyticus (Mli PAP). Using homology-based screening against E. coli PAP-1, we identified Mli PAP, sharing 54.8% sequence identity with its E. coli counterpart. The enzyme was expressed in E. coli but formed insoluble inclusion bodies; the active enzyme was purified as a fusion protein with the DsbA protein and used for functional assays. Mli PAP exhibited optimal activity at 30 °C and similar thermostability to E. coli PAP-1. ATP was the preferred substrate, with Km comparable to E. coli PAP-1 (1.61 mM and 1.70 mM, respectively), and Mg2+ (10 mM) was identified as the optimal cofactor. Mli PAP displayed salt-dependent activity, with the most effective polyadenylation in KCl and inhibition by NaCl and ammonium salts, contrasting with the halophilic nature of its host. This study provides the first functional insights into PAPs from Alphaproteobacteria, broadening the understanding of PAP diversity and biochemical properties, as well as the potential applications of PAPs in biotechnology.

Long interspersed nuclear elements-1 (LINEs-1) are known to be active human retrotransposons containing two protein-coding genes, ORF1 and ORF2, whose expression results in the production of two proteins-ORF1p-L1 and ORF2p-L1, respectively. Activation of LINE-1 may occur during the early stages of lung, prostate, esophageal, colon or breast cancer progression, often without obvious pathological symptoms or any overt signs of disease. In this study, we developed a method for preparing the LINE-1 proteins ORF1p-L1 and the reverse transcriptase (RT) domain of ORF2p-L1 and demonstrated their potential application as antigens in gastric cancer diagnostics. Both antigens were expressed as insoluble inclusion bodies in a bacterial expression system (E. coli BL21 (DE3) pLysS) using laboratory-scale fermentation. The preparation protocol involved solubilizing the inclusion bodies with a chaotropic agent, followed by multiple protein purification steps and subsequent renaturation of the proteins by dialysis under acidic conditions. At decreasing concentrations of the chaotropic agent, ORF1p-L1 and the RT domain of ORF2p-L1 were found to weakly bind to cation-exchange resins or to aggregate, whereas irreversible protein binding occurred under acidic conditions. The biological activity of the refolded ORF1p-L1 was confirmed by assessing its hybridization activity, while the reverse transcriptase activity of the RT domain of ORF2p-L1 was also successfully confirmed. Both refolded proteins reveal antigen-antibody response against antibodies in serum samples of patients with gastric cancer.

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF-mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF-mCherry did not bind Cu2+-charged IMAC resin, while moderate binding to Ni2+-charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production.

Errors in de novo synthesized DNA can originate from the oligonucleotides used during assembly. Oligonucleotides may contain substitutions, deletions, and insertions resulting from either incomplete reactions at individual steps of the phosphoramidite synthetic cycle or various side reactions. In this study, we quantitatively assessed errors in both gene constructs assembled from synthetic oligonucleotides by Sanger sequencing and in synthetic oligonucleotides by NGS. Our data demonstrate that side reactions involving carboxylic acid anhydrides during the capping step of oligonucleotide synthesis lead to the modification of guanine residues. This guanine modification subsequently results in the accumulation of G to A substitutions in the final gene constructs. We show that the error rate can be reduced by replacing the standard acetic anhydride-based capping mixture with anhydrides of carboxylic acids weaker than acetic acid. Furthermore, a more significant reduction in errors is achievable by using capping reagents based on phosphoramidite chemistry.

Background/Objectives: There is clear evidence for the higher prevalence of varicose veins (VVs) among women. In this regard, the research on sex differences affecting this condition is very important for sex-specific health care. We aimed to assess how male or female sex may contribute to the changes to gene expression profiles in the vein wall during varicose transformation. Methods: Paired varicose vein (VV) and non-varicose vein (NV) segments were harvested from patients with VVs after venous surgery. Processed RNAs from those samples were subjected to gene expression analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) followed by further data analysis. Multiple linear regression (MLR) analysis was performed to identify and characterize relationships among multiple factors (relative mRNA levels of a gene in NV or VV or their ratio, as dependent variables) and sex (independent variable, used individually or in combination with other patient’s characteristics). For sex-specific gene regulation analysis, all potential binding sites for sex hormone receptors were identified in each gene’s regulatory region sequence. Results: Using the independent method and a replicative patient sample set, we validated our previous data on 23 genes’ differential expression in VVs and obtained insights on their sex-specific regulation. Sex (as an individual independent variable or in combination with other parameters—patient characteristics such as Age, BMI, CEAP class, Height, VVD manifestation and duration) was a moderate predictor (0.40 < R < 0.59; p (R) < 0.05) for the STK38L expression in VVs (with its higher mRNA level in NVs and VVs of women compared to men); sex was a strong predictor (0.6 < R < 0.79; p (R) < 0.05) for the TIMP1 expression in VVs (with its lower mRNA level in VVs of women compared to men); sex was a moderate predictor (0.40 < R < 0.59; p (R) < 0.05) for the EBF1 expression in NVs (with its lower mRNA level in NVs of women compared to men). Conclusions: Confirmed differential expression of the studied genes in VVs indicates their plausible participation in vein wall remodeling. Sex-specific expression in veins for the subset of those genes suggests their hormonal regulation as well as other mechanisms involved in VV pathogenesis. This work enriches our understanding of sex features for the development of VVs and may provide the foundation for future investigations and beneficial treatment options.

Whooping cough, or pertussis, is a highly contagious disease caused by several Bordetella species and continues to pose a significant global public health concern. The rising incidence of pertussis highlights the urgent need for effective public health strategies to address Bordetella infections. Rapid and species-specific diagnostic tools are essential for preventing Bordetella transmission and are vital components of anti-infective measures. This study aimed to develop novel loop-mediated isothermal amplification (LAMP) and quantitative PCR (qPCR) assays for the detection of four Bordetella species responsible for human respiratory tract infections: B. pertussis, B. parapertussis, B. bronchiseptica, and B. holmesii. The qPCR assay demonstrated a low limit of detection (LoD), reliably identifying up to 5 copies of target DNA per reaction. The LAMP assays were approximately three times faster than qPCR (30 min) but had higher LoDs. Notably, qLAMP had a limit of detection of 25 copies per reaction for all four Bordetella species. In contrast, vLAMP had a LoD of 25 copies per reaction for B. pertussis and B. parapertussis; and a LoD of 50 copies per reaction for B. holmesii and B. bronchiseptica. We validated the assays using nasal swab samples from patients with respiratory tract infections, analyzing a total of 651 samples with qPCR and 145 samples with LAMP. Both assays exhibited no cross-reactivity with common viral and bacterial respiratory pathogens. The concordance rate between qPCR and LAMP was 94.5%, underscoring the reliability of both methods for clinical application. These findings suggest that the developed qPCR and LAMP tests can be successfully integrated into clinical practice for the detection and management of Bordetella infections.

The endothelium, as the inner layer of the vascular wall, is in constant contact with blood components, so that leukocytes have the ability to adhere to endotheliocytes and penetrate to the subendothelial space. When studying heterogenic vascular samples containing endothelial cells or pathological processes related to inflammation within the endothelium, it may be necessary to distinguish DNA by endothelial and leukocyte origin, which is possible due to its specific epigenetic modifications. To identify CpG loci that could serve as markers for endothelial cells, we searched for their distinctive stable methylated or demethylated states by applying marginal filtering (selecting CpG loci with methylation Beta values closer to 0 and 1) to the microarray data and identified 47 CpG loci with relatively stable methylation/demethylation status that differentiate endothelial (HUVEC, HCMEC, HPAEC, HPMEC, and LSEC) DNA from leukocyte (granulocytes, monocytes, and lymphocytes) DNA. In addition, we compared CpG loci with high and low levels of DNA methylation between different types of endothelial cells and leukocytes. We believe that the obtained data will hopefully facilitate further studies on endothelial dysfunction.

Poly(A) polymerase (PAP 1) from Escherichia coli is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP 1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, E. coli PAP 1’s toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP 1. For that, E. coli PAP 1 was expressed in seven E. coli strains with different origins and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP 1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18 °C) exacerbated PAP 1’s insolubility. Interestingly, PAP 1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker of PAP 1 activity. These findings provide insights into optimizing E. coli PAP 1 production for biotechnological applications.

The genetic underpinnings of elite sprint and power performance remain largely elusive. This study aimed to identify genetic variants associated with this complex trait as well as to understand their functional implications in elite sprint and power performance. We conducted a multi-phase genome-wide association study (GWAS) in world-class sprint and power athletes of West African and East Asian ancestry and their geographically matched controls. We carried out genotype imputation, replications for the top GWAS signal rs10196189 in two European cohorts, and gene-based and tissue-specific functional network analyses. For the first time, we uncovered the G-allele of rs10196189 in the Polypeptide N-Acetylgalactosaminyltransferase 13 (GALNT13) being significantly associated with elite sprint and power performance (P = 2.13E-09 across the three ancestral groups). Moreover, we found that GALNT13 expression level was positively associated with the relative area occupied by fast-twitch muscle fibers in the vastus lateralis muscle. In addition, significant and borderline associations were observed for BOP1, HSF1, STXBP2, GRM7, MPRIP, ZFYVE28, CERS4, and ADAMTS18 in cross-ancestry or ancestry-specific contexts, predominantly expressed in the nervous and hematopoietic systems. From the elite athlete cohorts, we further identified thirty-six previously uncharacterized genes linked to host defence, leukocyte migration, and cellular responses to interferon-gamma, and four genes - UQCRFS1, PTPN6, RALY and ZMYM4 - associated with aging, neurological conditions, and blood disorders. Taken together, these results provide new biological insights into the genetic basis of elite sprint and power performance and, importantly, offer valuable clues to the molecular mechanisms underlying elite athletic performance, health and disease.

The prevalence of insulin resistance (IR) is growing every year, which determines the risks of developing type 2 diabetes and cardiovascular diseases. Currently, IR is not recognized as a risk factor for the development of varicose veins (VVs), but the connection between the two is tacitly obvious because obesity and diabetes are risk factors for VVs. In this review, we have attempted to highlight the common nature of these two conditions in the context of mitochondrial dysfunction, inflammation, endothelial dysfunction, and tissue hypertrophy, and spotlight the role of IR in the development of VVs. We conclude that IR can contribute to the appearance of VVs.

Purpose: To evaluate the proportion of BRCA1 / 2 mutations in patients with serous and endometrioid cancer of the ovary, fallopian tube, and peritoneum in Russia, to evaluate the percentage of germinal and somatic mutations, to identify the spectrum of mutations in BRCA1 / 2 genes, to evaluate clinical and morphological features of the BRCA-associated ovarian cancer (OC).Patients and methods: The study enrolled patients of 18 years and older with newly diagnosed serous and endometrioid cancer of the ovary, fallopian tube, and peritoneum. Biological material (blood, tumor tissue) was collected, followed by molecular genetic analysis. The method of mutations detecting in the blood were: allele-specific PCR, high-resolution melting (HRM), Sanger sequencing method. Advanced genetic testing included the use of generation sequencing (NGS) and multiplex amplification of ligated probes (MLPA). The collection of clinical data, family history, clinical and morphological characteristics of the tumor was performed.Results: 500 patients were included in the study, the evaluation of BRCA1 / 2 mutations was performed in 496 patients (99,2 %). The frequency of BRCA1 / 2 mutations in the Russian patient population was 28,4 % (n = 141 / 496). The incidence of germinal mutations was 23,5 % (n = 117 / 141), and somatic — 4,8 % (n = 24 / 141). Frequent mutations in the Russian population were identified in 50 % of cases. When analyzing the ethnicity of patients in the Russian Federation BRCA-associated OC was most common in Russian (83,6 %, n = 118 / 141), Ukrainian (4,2 %, n = 6 / 141) and Tatar (3,5 %, n = 5 / 141) women. A family history of cancer was detected in 44 % of patients (n = 62 / 141) with BRCA1 / 2 mutations.Conclusions: Due to the high frequency of germinal and somatic BRCA1 / 2 mutations in the Russian patients it is recommended to conduct the advanced testing methods not only in blood samples but also in tumor tissue.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

The Beijing genotype is the most distributed M. tuberculosis family in Kazakhstan. In this study, we identified dominant Beijing clusters in Kazakhstan and assessed their drug susceptibility profiles and association with the most widely spread mutation Ser531Leu of the rpoB gene and the mutation Ser315Thr of the katG gene associated with resistance to rifampicin and isoniazid, respectively. M. tuberculosis isolates (n = 540) from new TB cases were included in the study. MIRU-VNTR genotyping was performed for 540 clinical isolates to determine M. tuberculosis families using 24 loci. RD analysis was additionally performed for the Beijing isolates. The identification of mutations in the drug-resistance genes of M. tuberculosis was performed with allele-specific real-time PCR and Sanger sequencing. The Beijing genotype was identified in 60% (324/540) of the clinical isolates. Central Asian/Russian cluster 94-32 was the most distributed cluster among the Beijing isolates (50.3%; 163/324). Three other dominant Beijing clusters were identified as 94-33 (3.4%; 11/324), 100-32 (3.1%; 10/324) and 99-32 (3.1%; 10/324). The Beijing genotype was associated with drug-resistant TB (p < 0.0001), including multidrug-resistant TB (p < 0.0001), in our study. An association of the mutation Ser531Leu of the rpoB gene with the Beijing genotype was found (p < 0.0001; OR = 16.0000; 95%CI: 4.9161–52.0740). Among the Beijing isolates, cluster 94-32 showed an association with MDR-TB (p = 0.021). This is why the evaluation of the Beijing genotype and its clusters is needed to control MDR-TB in Kazakhstan.

The purpose of the study was to compare and analyze the levels of KREC (kappa-deleting recombination excision circle) and TREC (T cell receptor excision circle) levels, which indirectly reflect impaired maturation of T and/or B lymphocytes, in children of the medical and biological risk group and the comparison group (patients considered relatively healthy, relative to the population).The medical and biological risk groups were:1) 15 children with operated congenital heart defects with combined thymectomy and 9 without it; the average age is 5 months ±4 months and 7 months ±3 months, respectively; and2) 27 children with relatively frequent morbidity of the respiratory tract (acute respiratory viral infection more than 8 times a year) aged 1.6±1.4 months, among whom the proportion attending a preschool institution is 20 people (74%).The comparison groups were:1) 16 relatively healthy children (health group 1) aged 1.7±1.6 years, among whom the proportion of those attending a preschool institution was 13 people (81%); and2) 48 apparently healthy newborn children, whose average age was 15±12 days.Quantitative determination of TREC and KREC was carried out using a multiplex test system developed at the Institute of Chemical Biology and Fundamental Medicine of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). The average concentrations of TREC and KREC were determined in apparently healthy newborns. Children with a high incidence of infectious respiratory morbidity (more than 8 cases of acute respiratory viral infection per year) had a significantly higher risk of developing disorders of both T cell and B cell immunity, compared with the healthy population. Children who underwent total thymectomy during surgical treatment of congenital heart defects had a higher risk of developing immunodeficiency conditions, affecting predominantly T cell immunity, in comparison with the group of children operated on with preservation of the thymus gland.

Making a correct genetically based diagnosis in patients with diseases associated with mitochondrial dysfunction can be challenging both genetically and clinically, as can further management of such patients on the basis of molecular-genetic data assessing the state of their mitochondria. In this opinion article, we propose a novel approach (which may result in a clinical protocol) to the use of a precise molecular-genetic tool in order to monitor the state of mitochondria (which reflects their function) during treatment of certain conditions, by means of not only signs and symptoms but also the molecular-genetic basis of the current condition. This is an example of application of personalized genomic medicine at the intersection of a person’s mitochondrial genome information and clinical care. Advantages of the proposed approach are its relatively low cost (compared to various types of sequencing), an ability to use samples with a low input amount of genetic material, and rapidness. When this approach receives positive outside reviews and gets an approval of experts in the field (in terms of the standards), it may then be picked up by other developers and introduced into clinical practice.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

Human adenoviruses (HAdVs) are common pathogens that are associated with a variety of diseases, including respiratory tract infections (RTIs). Without reliable, fast, and cost-effective detection methods for HAdVs, patients may be misdiagnosed and inappropriately treated. To address this problem, we have developed a multiplex loop-mediated isothermal amplification (LAMP) assay for the detection of the species Human adenovirus B (HAdV-B), Human adenovirus C (HAdV-C) and Human adenovirus E (HAdV-E) that cause RTIs. This multiplexing approach is based on the melting curve analysis of the amplicons with a specific melting temperature for each HAdV species. Without the need for typing of HAdVs, the LAMP results can be visually detected using colorimetric analysis. The assay reliably detects at least 375 copies of HAdV-B and -C and 750 copies of HAdV-E DNA per reaction in less than 35 min at 60 °C. The designed primers have no in silico cross-reactivity with other human respiratory pathogens. Validation on 331 nasal swab samples taken from patients with RTIs showed a 90–94% agreement rate with our in-house multiplex quantitative polymerase chain reaction (qPCR) method. Concordance between the quantitative and visual LAMP was 99%. The novel multiplexed LAMP could be an alternative to PCR for diagnostic purposes, saving personnel and equipment time, or could be used for point-of-care testing.

Background and aims DiGeorge syndrome (DGS) is a genetic disorder manifesting in polymorphic symptoms related to developmental abnormalities of various organs including thymus. DGS is caused by microdeletions in the 22q11.2 region between several low copy repeats (LCR) occurring in approximately 1 in 4000 live births. Diagnosis of DGS relies on phenotypic examination, qPCR, ultrasound, FISH, MLPA and NGS which can be relatively inaccurate, time-consuming, and costly. Materials and methods A novel multiplex droplet digital PCR (ddPCR) assay was designed, optimized and validated for detection and mapping 22q11.2 microdeletions by simultaneous amplification of three targets — TUPLE1, ZNF74, D22S936 — within the deletion areas and one reference target — RPP30 — as an internal control. Results The assay reliable identified microdeletions when the template concentration was >32 copies per reaction and successfully detected LCR22A-B, LCR22A-C, LCR22A-D, and LCR22B-C deletions in clinical samples from 153 patients with signs of immunodeficiency. In patients with the microdeletions, flow cytometry detected a significant increase in B-cell and natural killer cell counts and percentages, while T-cell percentages and T-cell receptor excision circle (TREC) numbers decreased. Conclusion The designed ddPCR assay is suitable for diagnosing DGS using whole blood and blood spots.