Restriction Fragment Length Polymorphism Analysis Research Articles

Efficacy of CLT4A variants as immunoregulatory molecules among Vitiligo patients in Saudi Arabia

Vitiligo is a skin depigmentation condition caused by the immune-mediated perdition of melanocytes. In vitiligo, cytotoxic T-lymphocytes damage melanocytes, causing skin depigmentation. The Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) gene has been reported to be related to vitiligo and other autoimmune diseases. The purpose of this study was to explore the molecular involvement of rs231775 and rs3087243 SNPs in the CTLA4 gene in vitiligo patients. The recruitment was based on the sample size calculation and based on it, we have recruited 300 Saudi individuals who were evenly divided into vitiligo cases and controls. Extracted genomic DNA was utilized to amplify rs231775 and rs3087243 SNPs in the CTLA4 gene, which were subsequently digested and verified using Sanger sequencing. The polymerase chain reaction and restriction fragment length polymorphism analysis laboratory data were stored in Excel and used for further statistical analysis. Although baseline details had no correlation (p > 0.05) between the two groups, Hardy-Weinberg Equilibrium analysis was in agreement with the vitiligo patients (p > 0.05). The genotype and allele frequencies were frequently associated (p < 0.05) with rs3087243, and rs231775 SNP showed a nominal association with GG genotype and G allele (p < 0.05). The specific relationship in vitiligo cases was revealed by MDR, GDMR, and linkage disequilibrium (p < 0.05), but not with haplotype analysis (p > 0.05). Thus, the study concluded that rs3087243 SNP was associated and rs231775 SNP showed a nominal association with vitiligo in the Saudi population.

Association Between the Aromatase rs2414096 Polymorphism and Prostate Cancer

Abstract Objectives The purpose of this study was to investigate association between the aromatase (CYP19A1) rs2414096 polymorphism and the risk of prostate cancer development, progression and aggressiveness in the Slovak population. Background Aromatase catalyses the final reaction of androgens aromatization to estrogens, irreversible conversion of androstenedione to estrone and testosterone to estradiol. Methods The study population consisted of 721 prostate cancer patients and 660 men in the control group. Genotyping was performed via restriction fragment length polymorphism analysis. The expression levels of CYP19A1 in prostate cancer tissues were compared with those in benign prostatic hyperplasia tissues. Results The CYP19A1 rs2414096 AA genotype and A allele were significantly associated with an increased risk of prostate cancer development. The significant association between this genotype and PSA level ≥ 10 ng/ml, Gleason score ≥ 7 and with the presence of metastases was observed. Significantly decreased relative expression of CYP19A1 was detected in prostate cancer tissues. Patients with the AA genotype presented higher relative CYP19A1 expression than did those with the GG genotype. Significantly lower circulating testosterone levels and higher serum PSA levels were detected in patients with the AA genotype. Conclusion The CYP19A1 rs2414096 polymorphism was significantly associated with prostate cancer development and aggressiveness in Slovak patients.

Characteristics of polymorphism of genes involved in the regulation of glucose metabolism and steroid hormone synthesis in patients with polycystic ovary syndrome

Background: Polycystic ovary syndrome is an urgent problem of gynecological endocrinology. Currently, a number of genes have been studied that control glucose metabolism and are involved in the synthesis, conversion into an active form and transport of steroid hormones, mutations in which with a certain degree of reliability can serve as a diagnostic criterion for polycystic ovary syndrome. Aim: The aim of this study was to evaluate the PPARG Pro12Ala, INS 223HphI AT, and SHBG (TAAAA)n gene polymorphisms in patients with polycystic ovary syndrome and in healthy individuals. Materials and methods: The polymerase chain reaction method and restriction fragment length polymorphism analysis were used. The frequencies of alleles and genotypes of polymorphic variants were studied in 136 women with polycystic ovary syndrome and 47 women in the control group: Pro12Ala (PPARG gene), 223HphI AT, (INS gene) and (TAAAA)n repeats (SHBG gene). Results: The distribution of the allele and genotype frequencies for the PPARG (rs1801282) and INS (rs689) genes in patients with polycystic ovary syndrome and in healthy individuals did not differ. The distribution of the genotype frequencies in the group of women with polycystic ovary syndrome differed (p = 0.02) from that in the control group for the (TAAAA)n polymorphism in the SHBG gene. In the presence of a long repeat in the SHBG gene in at least one of the homologous chromosomes, the probability of a woman having polycystic ovary syndrome increases by 2.5 times. Patients with a verified A/A genotype (INS), in the presence of an SHBG gene allele with a long repeat, have a ten-fold higher risk of developing polycystic ovary syndrome than women with SHBG gene short alleles. Conclusions: Patients with long repeats in the SHBG gene are at risk for developing polycystic ovary syndrome with phenotypes A, B, and C, especially in combination with the presence of the A/A genotype, class I (INS).

Association of rs3755319 and rs4148325 of the &lt;i&gt;UGT1A1&lt;/i&gt; gene, rs2328136 of the &lt;i&gt;NUP153-AS&lt;/i&gt; gene, and rs16928809 of the &lt;i&gt;SLC22A18&lt;/i&gt; gene with benign unconjugated hyperbilirubinemia

Background: Benign unconjugated hyperbilirubinemia, also known as Gilbert's syndrome, is a common in the population moderate increase in total and unconjugated bilirubin concentrations in individuals without liver disease or hemolysis. Aim: To identify associations of rs3755319, rs4148325 of the UGT1A1 gene, rs2328136 of the NUP153-AS gene, and rs16928809 of the SLC22A18 gene with benign unconjugated hyperbilirubinemia. Methods: This case-control study included a group of individuals with benign unconjugated hyperbilirubinemia (n = 414, mean age 36.7 ± 15.9 years, 49.8% men) and a control group (n = 381, mean age 39.1 ± 15.9 years, 52.5% men). The sample was randomly selected from the participants of the MONICA project, screening of young people aged 25–44 years and a cross-sectional study of schoolchildren in Novosibirsk. DNA was isolated from venous blood by phenol-chloroform extraction or an express assay (PROBA-RAPID-GENETICS, DNA-Technology, Russia). Genotyping of the groups by rs3755319, rs4148325 of the UGT1A1 gene, rs2328136 of the NUP153-AS gene, and rs16928809 of the SLC22A18 gene was performed by polymerase chain reaction followed by restriction fragment length polymorphism analysis. Results: No significant differences were found between the individuals with benign unconjugated hyperbilirubinemia and the control group by the genotypes and alleles of the nucleotide sequence variants of rs16928809 of the SLC22A18 gene (p 0.05). The CC genotype and the C allele of rs3755319 of the UGT1A1 gene were more common in the individuals with benign unconjugated hyperbilirubinemia, than in the control group (CC vs AC + AA: odds ratio (OR) = 21.1, 95% confidence interval (CI) 14.7–30.4, p 0.001; C vs A: OR = 12.4, 95% CI 9.4–16.4, p 0.001). The concentrations of total and unconjugated bilirubin were higher in the carriers of the CC genotype of rs3755319, compared to the carriers of the other two genotypes (p 0.05). rs4148325 of the UGT1A1 gene was in the linkage disequilibrium with the rs3064744 UGT1A1 variant. The GG genotype and the G allele rs2328136 of the NUP153-AS gene were more common in the individuals with benign unconjugated hyperbilirubinemia than in the control group (GG vs AG + AA: OR = 1.361, 95% CI 1.002–1.848, p = 0.048; G vs A: OR = 1.33, 95% CI 1.02–1.73, p = 0.034). Conclusion: The CC genotype and the C allele of rs3755319 of the UGT1A1 gene, the GG genotype and the G allele of rs2328136 of the NUP153-AS gene are the genotypes and alleles of risk for benign unconjugated hyperbilirubinemia. The rs16928809 of the SLC22A18 gene is not associated with benign unconjugated hyperbilirubinemia.

Effects of Hinc II Polymorphism in the LDL Receptor Gene on Serum Lipid Levels of Jordanian Individuals with High Risk for Coronary Heart Disease

Coronary heart disease (CHD) has presented high prevalence in the Jordanian population. Nevertheless, studies of genetic risk factors for CHD in our country are insufficiently carried out. We are intended to investigated the effects of Hinc II (exon 12) polymorphisms at the low-density lipoprotein receptor (LDLR) gene on circulating lipids of 150 individuals with high risk for CHD (HRG) and 150 controls (C). Genomic DNA was extracted from white blood cells and amplified by polymerase chain reaction (PCR) and digested with HincII Restriction enzyme. LDL, TC (total cholesterol), TG (triglycerides), and HDL (high density lipoproteins) levels were measured in all subjects. RFLP (restriction fragment length polymorphism) analysis was conducted to identify genotype of LDLR gene in (HRG) and 150 controls (C). The results showed a significant correlation existed between this RFLP locus and (HRG), X2=10.6, P&amp;lt;0.05; H&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt; allele frequency: X2=7.88, P&amp;lt;0.05., H&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt; allele frequency: X2=7.88, P&amp;lt;0.05. Genotypes H&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt;H&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt; and H&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt;H&amp;lt;sup&amp;gt;- &amp;lt;/sup&amp;gt;in high risk group are significantly associated with high levels of LDL, TC, TG, P&amp;lt;0.05 and with low level of HDL P&amp;lt;0.05., while H&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt;H&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt; is associated with normal values of serum lipids P&amp;lt;0.05. It is inferred that H&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt; allele might be associated with high blood cholesterol level, and the H&amp;lt;sup&amp;gt;-&amp;lt;/sup&amp;gt; allele with normal level. This study suggests that the differences in LDLRG genotypes might affect the phenotype of lipid metabolism.

Novel Findings on the SNP18 Sequence and Its Functional Analysis in Hygienic Behavior of Apis mellifera.

Hygienic behavior (HB) is a crucial biological mechanism in honeybees that is associated with disease resistance. This study aimed to investigate the effect of the SNP18 sequence and environmental factors on the HB of honey bees, using a total of 14 colonies and 148 bee samples from seven different bee breeds. Association analysis revealed that colonies with Italian hybrids (IH) or young queens statistically (p < 0.01) exhibited high hygienic behavior (HHB). HB increased by 71.6% when the number of frames in the colony, representing colony power (CP), increased by one (p < 0.05). In restriction fragment length polymorphism (RFLP) analysis, novel mutations in the MlyI polymorphism of the SNP18 sequence were firstly found in Apis mellifera. In addition, the restriction fragments of the novel variants of the SNP18 HHB and SNP18 low hygienic behavior (LHB) lines were determined by sequencing. In this study, several important findings emerged: Due to one-base differences in the restriction fragment, this sequence could not be genotyped by RFLP. Honeybees could be homozygous (HHB or LHB) or heterozygous (HHB and LHB) for the SNP18. SNP18 sequence could be located in different regions of the chromosome and could only be determined by genome sequencing. Finally, since genotypes cannot be clearly determined, no specific allele or genotype can be recommended for HB selection in beekeeping. Therefore, additional research is required to assess discovered novel variants for genetic selection of HHB for ecological beekeeping, healthy products and sustainability.

Engineered FnCas9 mediated mutation profiling for clarithromycin resistance in Helicobacter pylori strains isolated from Indian patients with gastrointestinal disorders

Helicobacter pylori is a highly prevalent gut pathogen with reported implications in a wide range of gastrointestinal disorders. Antibiotic based therapy, especially with clarithromycin is one of most effective treatment strategies against H. pylori. However, rising global prevalence of clarithromycin resistance in certain H. pylori strains, primarily attributed to point mutations in the 23 s ribosomal RNA coding gene, pose a major challenge in the effective eradication of this pathogen. There are a number of established methodologies devised for H. pylori mutation detection, so as to provide a tailored treatment plan to the patients and resist further transmissions of antibiotic resistant strains. However, there is no ‘gold standard’ method available to detect mutation status in clinical isolates of H. pylori from infected patients. CRISPR-Cas9 based technologies have revolutionized the field of mutation detection in biological samples, particularly during the recent COVID-19 pandemic. Although multiple assays have been reported for detection of H. pylori in clinical samples including CRISPR diagnostics (CRISPRDx) platforms, there is no such assay reported till date to detect specific mutations that confer antibiotic resistance to this pathogen. In this study, we have developed an assay using engineered FnCas9 (en31-FnCas9) protein to effectively detect the A2142G and A2143G mutations in the 23 s rDNA of H. pylori strains isolated from gastric biopsy samples. The data from in vitro cleavage assays and strip-based lateral flow tests using en31-FnCas9 and guide RNAs targeting the conserved and mutated loci of H. pylori 23 s rDNA are in perfect congruence with the data from Sanger sequencing and restriction fragment length polymorphism (RFLP) analysis. Our results indicate that en31-FnCas9 based mutation analysis can be deployed as an efficient diagnostic methodology to detect clarithromycin susceptibility in patients with suspected H. pylori infections.

Genetic Polymorphisms of DNA Repair Genes and their Influence on Paclitaxel based Chemotherapy Induced Toxicity Reactions in Breast Cancer Patients.

Systemic chemotherapy constitutes an indispensable component of breast cancer (BC) management, where therapeutic drug combinations such as anthracyclines, platinum compounds, and taxanes form the cornerstone of standard treatment protocols. Although DNA repair genes are pivotal in cancer susceptibility, their specific roles in mediating acute or chronic toxicity outcomes induced by chemotherapy remain undetermined. Consequently, this study was planned to elucidate the impact of polymorphisms in base excision repair (BER) genes, including XRCC1, XRCC2, XRCC3, APE1, and hOGG1, on treatment response and toxicity outcomes in BC patients undergoing paclitaxel and doxorubicin-based chemotherapy within an Indian population. One hundred and four (104) BC patients receiving combined paclitaxel and doxorubicin chemotherapy were enrolled with documentation of both hematological and non-hematological toxicity reactions induced by the treatment. Genetic polymorphism of XRCC1, XRCC2, XRCC3, APE1, and hOGG1 genes was investigated using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) analysis. Analysis of the demographic characteristics of BC patients revealed a significant association between mucositis and peripheral neuropathy with advancing age. An increased body mass index was also significantly correlated with hematological toxicities, such as neutropenia (p=0.022) and febrile neutropenia (p=0.048), as well as with peripheral neuropathy (p=0.001). Univariate logistic regression analysis demonstrated a significant association between the XRCC3 (Ser241Cys) polymorphism and peripheral neuropathy (OR=3.00, 95% CI: 1.29-6.95; p=0.010). Similarly, regression analysis indicated a significant association of APE-1 (Asp148Glu) polymorphism with febrile neutropenia (OR=3.55, 95% CI: 1.03-12.21; p=0.044) and chemotherapy-induced nausea and vomiting (CINV) (OR=4.19, 95% CI: 1.61-10.94; p=0.003) in BC patients treated with paclitaxel and Doxorubicin regimen. The findings from this study underscore the significant influence of genetic polymorphisms in XRCC3 (Ser241Cys) and APE-1 (Asp148Glu) on the acute toxicity effects induced by paclitaxel in BC patients.

Diagnostic Implications of CD63 and CD64 Expression Levels and FcγRIIIA 158 V/F Gene Polymorphism in Primary Immune Thrombocytopenia Adult Patients.

Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disease characterized by reduced platelet counts due to immune system dysregulation caused by many factors, including genetics, autoimmune diseases, infections, and inflammations. Therefore, the current study aimed to evaluate immunological markers such as the expression level of lysosomal associated membrane protein 3 (LAMP-3), also known as CD63, and the expression level of Fc-gamma receptor I (FcγRI), also known as CD64 and also investigate the association of Fc-gamma receptor IIIA (FcγRIIIA) 158 V/F polymorphism to the risk of ITP. A total of 180 subjects; 60 ITP patients, 60 patients with thrombocytopenia of other causes and 60 controls were enrolled into our study. The expression level of CD63 was done using reverse transcription quantitative PCR (RTqPCR), while CD64 expression level was done by flow cytometry. The polymorphism of FcγRIIIA 158 V/F gene was analyzed by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis. Finally, CD63 and CD64 protein-protein interactions were done by using the STRING online database. The expression of CD63 was significantly elevated in ITP patients than thrombocytopenia patients and healthy control. Also there was high expression level of CD64 on granulocytes and monocytes from ITP patients than other groups. Receiver operating characteristic curve (ROC curve) analysis of CD63 showed an area under the curve (AUC) revealed of 1.00, sensitivity of 100% and specificity of 100%; while for CD64 on granulocytes, AUC of 0.998 as well as a sensitivity of 96.66% and specificity of 93.33%. Regarding FcγRIIIa 158 V/F polymorphism, all patients and healthy volunteers included in this study showed the wild FF genotype. The expression of both CD63 and CD64 were significantly increased in ITP patients and could be good biomarkers to diagnose ITP. Additionally, there is no association between FcγRIIIa 158 V/F polymorphism and the risk of ITP disease.

Molecular analysis of vector-borne pathogens in Eurasian badgers (Meles meles) from continental Europe

BackgroundVector-borne pathogens (VBPs) are increasing in significance in veterinary medicine and public health settings, with wildlife playing a potentially crucial role in their transmission. Eurasian badgers (Meles meles) are widely distributed across Europe. However, information currently available on the prevalence of VBPs in badgers is limited. The objective of the current study was to investigate the occurrence of Anaplasmataceae, Bartonella spp., Mycoplasma spp., Rickettsia spp., Piroplasmida, Trypanosomatida and Filarioidea in badgers and subsequently, based on the results, assess the potential risk to domestic animals, other wildlife and humans.MethodsBetween 2017 and 2021, blood or spleen samples from 220 badgers were collected in nine continental European countries: Austria (n = 7), Bosnia and Herzegovina (n = 2), Croatia (n = 22), France (n = 44), Germany (n = 16), Hungary (n = 7), Italy (n = 16), Romania (n = 80) and Serbia (n = 26). VBPs were identified by performing PCR analysis on the samples, followed by Sanger sequencing. Additionally, to distinguish between different Babesia lineages we performed restriction fragment length polymorphism (RFLP) analysis on piroplasm-positive samples, using HinfI as restriction enzyme. A phylogenetic analysis was performed on Mycoplasma spp.ResultsThe pathogens identified were Babesia sp. badger type A (54%), B (23%), and C (37%); Trypanosoma pestanai (56%); Mycoplasma sp. (34%); Candidatus Mycoplasma haematomelis (8%); Candidatus Mycoplasma haematominutum (0.5%); and Ehrlichia spp. (2%). Rickettsia spp., Bartonella spp. and filarioid nematodes were not detected among the tested samples.ConclusionsThe large sample size and diverse study populations in this study provide valuable insights into the distribution and epidemiology of the analyzed pathogens. Some of the VBPs identified in our study show high similarity to those found in domestic animals, such as dogs. This finding suggests that badgers, as potential reservoirs for these pathogens, may pose a threat not only to other wildlife but also to domestic animals in close vicinity. Continuous surveillance is essential to monitor VBPs in wildlife as a means to enable the assessment of their impact on other wildlife species, domestic animals and human health.Graphical

Molecular detection of Leishmania DNA in wild-caught sand flies, Phlebotomus and Sergentomyia spp. in northern Iran

Leishmaniasis is currently considered a major health problem in Iran, posing an increasing threat to society's development in various dimensions. This study aimed to detect Leishmania infection in wild-caught sand flies in Sari City, northern Iran. Sand flies were collected using sticky traps, and Leishmania DNA was identified using polymerase chain reaction (PCR) targeting the ITS2-rDNA region, followed by restriction fragment length polymorphism (RFLP) analysis. A total of 138 female sand flies were tested, among which, only 1 specimen of Ph. papatasi (11.11 %) and Ph. major (14.28 %), 4 specimens of Ph. kandelakii (7.27 %) and Se. dentata (8.33 %), and 2 specimens of Se. sintoni (50 %) were naturally infected with L. (L.) major. This was observed in the ITS2 nested-PCR amplification assays where a ∼ 245 bp PCR band was produced. Also, RFLP analysis by Mnl1 revealed the fragments of 55 and 70 and 120 bp for infected sand flies which are characteristic of L. (L.) major. Most of the sand flies were unfed, collected during warm season, found indoor. This study reperesents the first molecular detection of L. (L.) major in wild-caught sand flies, specifically in Ph. papatasi in this region, as well as Ph. kandelakii and Ph. major in Iran and even the world.

Effects of APOE isoforms in diabetic nephropathy patients of South India.

Diabetic nephropathy (DN) is a grave complication and the most common renal dysfunction of diabetes mellitus. Genetic factors, including Apolipoprotein E (APOE) isoforms, have been implicated in the pathogenesis of DN. A total of 577 type 2 Diabetes mellitus subjects were categorized into diabetes non-nephropathic (Controls: n = 321), diabetes nephropathic (DN: n = 256) groups. Demographic, clinical, and biochemical parameters including age, BMI, lipid profiles (TC, LDL-C, HDL-C, TG), glucose metabolism (plasma glucose, HbA1c, serum insulin), renal function (UACR, PCR), and blood pressure (SBP, DBP) were assessed. APOE variant frequencies were determined using restriction fragment length polymorphism (RFLP) analysis, validated against Hardy-Weinberg equilibrium (HWE), and statistically correlated with each clinical and biochemical parameter. The DN group had an increased prevalence of hypertension, fatty liver, and dyslipidemia compared to the Control group. Biochemical analyses revealed elevated levels of TC (213.41mg/dL vs. 189.32mg/dL), LDL-C (134.46mg/dL vs. 107.56mg/dL), and reduced HDL-C (58.13mg/dL vs. 65.32mg/dL) in DN cases compared to Controls (all p < 0.0001). The APOE variants distribution showed a significant increase in E2 allele frequency (69.1% vs. 15.3%) and corresponding homozygous genotype (E2/2: 42.2% vs. 5.6%) in DN cohorts. The study found a higher frequency of E2 allele in the DN group compared to Controls, though no statistically significant risk of DN was linked to this allele. The results suggest a potential association for APOE polymorphisms, requiring broader studies to clarify the role of APOE polymorphisms in DN susceptibility.

An Investigation of Polymorphism on the FASN, SCD1, and SLC27A3 Genes in Sheep

Milk traits of sheep are affected by many environmental and genetic factors. These traits are quantitative traits and are determined by many genes. This study aimed to investigate polymorphisms of milk-related Fatty-acid synthase (FASN), Stearoyl-CoA desaturase (SCD1), and solute carrier 27A3 (SLC27A3) genes in cross-bred Hamdani sheep. Blood samples were collected from 100 healthy cross-bred Hamdani ewes from the jugular vein into K3-EDTA-containing tubes. Genomic DNA was extracted using a commercial DNA isolation kit. PCR products of FASN (275 bp), SCD1 (225 bp), SLC27A3-P1 (341 bp), and SLC27A3-P2 (319 bp) were subjected to restriction fragment length polymorphism (RFLP) analysis using SSil (AciI), Cfr13I, AluI, and Hpy188III the restriction enzyme, respectively. However, all gene regions were found to be monomorphic. In the study, only TT, AA, AA, and GG genotypes were detected for FASN, SCD1, SLC27A3-P1, and SLC27A3-P2, respectively. Allele and genotype frequencies were 1.00 for all genotypes and alleles. Although this study did not reveal favorable genotypes in FASN, SCD1, and SLC27A3 genes that can be used for milk traits, more comprehensive studies with larger sample sizes should be conducted to investigate polymorphisms in cross-bred Hamdani sheep.

Genetic characterization of Neisseria meningitidis isolates recovered from patients with invasive meningococcal disease in Lithuania.

Neisseria meningitidis is a gram-negative bacterium responsible for life-threatening invasive infections known as invasive meningococcal disease and is associated with high fatality rates and serious lifelong disabilities among survivors. This study aimed to characterize N. meningitidis isolates cultured from blood and cerebrospinal fluid collected between 2009 and 2021 in Lithuania, assess their genomic relationships with European strains, and evaluate the possibility of using a cost-effective method for strain characterization, thus improving the national molecular surveillance of invasive meningococcal disease. In total, 321 N. meningitidis isolates were collected and analyzed using multilocus restriction typing (MLRT). Amplification of the penA gene and restriction fragment length polymorphism analysis were performed to identify the modified penA genes. Based on the MLRT genotyping results, we selected 10 strains for additional analysis using whole-genome sequencing. The sequenced genomes were incorporated into a dataset of publicly available N. meningitidis genomes to evaluate genomic diversity and establish phylogenetic relationships within the Lithuanian and European circulating strains. We identified 83 different strains using MLRT genotyping. Genomic diversity of N. meningitidis genomes analysed revealed 21 different sequence types (STs) circulating in Lithuania. Among these, ST34 was the most prevalent. Notably, three isolates displayed unique combinations of seven housekeeping genes and were identified as novel STs: ST16969, ST16901, and ST16959. The analyzed strains were found to possess virulence factors not commonly found in N. meningitidis. Six distinct penA profiles were identified, each with different frequencies. In the present study, we also identified N. meningitidis strains with new penA, NEIS0123, NEIS1320, NEIS1525, NEIS1600, and NEIS1753 loci variants. In our study, using the cgMLST scheme, Minimum Spanning Tree (MST) analysis did not identify significant geographic relationships between Lithuanian N.meningitidis isolates and strains from Europe. Discussion: To our knowledge, this is the first study to employ whole genome sequencing (WGS) method for a comprehensive genetic characterization of invasive N. meningitidis isolates from Lithuania. This approach provides a more detailed and precise analysis of genomic relationships and diversity compared to prior studies relying on traditional molecular typing methods and antigen analysis.

Effects of Fallow Season Water and Straw Management on Methane Emissions and Associated Microorganisms

The effects of fallow season water and straw management on methane (CH4) emissions during the fallow season and the subsequent rice-growing season are rarely reported, and the underlying microbial mechanisms remain unclear. A field experiment was conducted with four treatments: (1) fields flooded in both the fallow and rice seasons (FF), (2) fields drained in the fallow season and flooded in the rice season (DF), (3) FF with straw retention (FFS), and (4) DF with straw retention (DFS). The CH4 emissions in fields under different water and straw treatments were monitored using the static closed chamber method. Methanogenic and methanotrophic communities in these fields were examined using terminal restriction fragment length polymorphism (T-RFLP) analysis based on the mcrA gene and pmoA gene encoding methyl coenzyme M reductase and particulate methane monooxygenase, respectively. The results showed that CH4 emissions were significantly affected by water management, straw retention, season, and their interactions. Over 80% of CH4 emissions occurred during the rice season. Field drainage during the fallow season reduced CH4 emissions by 47.0% and 53.8% with and without straw during the rice season, respectively. Water management altered the abundance and composition of methanogens and methanotrophs, whereas the effects of straw retention were less pronounced. The quantitative polymerase chain reaction (qPCR) assay revealed that field drainage in the fallow season decreased the mcrA gene abundance by 30.0% and 23.2% with and without straw in rice season, respectively, and increased the pmoA gene abundance by 108.9% and 213.7% with and without straw in rice season, respectively. CH4 flux was significantly positively associated with mcrA gene copy number and the ratio of mcrA to pmoA gene copy number, whereas it was significantly negatively correlated with the pmoA gene copy number. Results indicated that fallow drainage greatly decreased CH4 emission not only during the fallow season but also during the subsequent rice season by altering the community composition of methanogens and methanotrophs. These findings provide scientific insight into the role of water and straw management in controlling CH4 emissions through microbial community dynamics.

Molecular genetic methods for quality control of inactivated vaccines using a Chikungunya virus model: vaccine strain identification and completeness of virus inactivation

INTRODUCTION. The completeness of virus inactivation and the identity of the vaccine strain are essential parameters for the safety and quality of inactivated virus vaccines, which should be controlled during vaccine development and production. Currently, the most promising quality control methods for inactivated virus vaccines are molecular genetic methods, which provide rapid results with high sensitivity and specificity.AIM. The aim of this study was the development of a real-time quantitative polymerase chain reaction (qPCR) method and an integrated cell culture real-time quantitative polymerase chain reaction (ICC-qPCR) method to assess the completeness of virus inactivation, as well as a reverse-transcription polymerase chain reaction assay coupled with restriction fragment length polymorphism analysis (RT-PCR-RFLP) to confirm the identity of the vaccine virus strain.MATERIALS AND METHODS. This study used RNA of CHIKV genotypes (three strains of each of the four CHIKV genotypes, including Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL), which were identified by sequencing prior to analysis). Additionally, the study used the Nika21 CHIKV strain (ECSA genotype), the Nika21 CHIKV strain inactivated with β-propiolactone, and the Nika21 CHIKV strain antigen adsorbed on aluminium hydroxide. The methods used included real-time qPCR, RT-PCR-RFLP, and virus neutralisation.RESULTS. The study identified a 218 bp fragment of the nsP1 gene (positions 789 to 1006) with restriction endonuclease recognition sites. These sites were present or absent in combinations specific to each of the four CHIKV genotypes. The authors selected primers for amplification of the specified gene region and tested the conditions for real-time qPCR and RT-PCR-RFLP. The study demonstrated the possibility of using the ICC-qPCR method to confirm the completeness of virus inactivation and the RT-PCR-RFLP method to identify the vaccine strain.CONCLUSIONS. The study showed the advantages of using the ICC-qPCR method to confirm the completeness of antigen inactivation and the RT-PCR-RFLP method to identify the vaccine strain. These methods are more sensitive and faster than traditional culture methods. ICC-qPCR and RT-PCR-RFLP can be used at any stage of the production process for inactivated vaccines.

The role of TiO2 and gC3N4 bimetallic catalysts in boosting antibiotic resistance gene removal through photocatalyst assisted peroxone process

Antibiotics are extensively used in human medicine, aquaculture, and animal husbandry, leading to the release of antimicrobial resistance into the environment. This contributes to the rapid spread of antibiotic-resistant genes (ARGs), posing a significant threat to human health and aquatic ecosystems. Conventional wastewater treatment methods often fail to eliminate ARGs, prompting the adoption of advanced oxidation processes (AOPs) to address this growing risk. The study investigates the efficacy of visible light-driven photocatalytic systems utilizing two catalyst types (TiO2-Pd/Cu and g-C3N4-Pd/Cu), with a particular emphasis on their effectiveness in eliminating blaTEM, ermB, qnrS, tetM. intl1, 16 S rDNA and 23 S rDNA through photocatalytic ozonation and peroxone processes. Incorporating O3 into photocatalytic processes significantly enhances target removal efficiency, with the photocatalyst-assisted peroxone process emerging as the most effective AOP. The reemergence of targeted contaminants following treatment highlights the pivotal importance of AOPs and the meticulous selection of catalysts in ensuring sustained treatment efficacy. Furthermore, Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analysis reveals challenges in eradicating GC-rich bacteria with TiO2 and g-C3N4 processes, while slight differences in Cu/Pd loadings suggest g-C3N4-based ozonation improved antibacterial effectiveness. Terminal Restriction Fragment Length Polymorphism analysis highlights the efficacy of the photocatalyst-assisted peroxone process in treating diverse samples.

Molecular characterization of ‘Candidatus Phytoplasma australasiaticum’ associated with ornamentals, amaranthus, French bean and sunhemp crops and development of rapid LAMP assay for detection

Phytoplasma is one of the most economically important pathogens, causing significant yield losses in many crops worldwide. During a survey in Bengaluru rural and Chikkabalpura areas of Karnataka, India, a total of 33 symptomatic plant samples including ornamentals, amaranthus, french bean, and sunhemp displaying phytoplasma symptoms were collected and confirmed as infected with phytoplasma through PCR targeting the 16S rRNA and secY genes. Pairwise sequence comparison and phylogenetic analysis of 16S rRNA and secY gene sequences confirmed the presence of ‘Candidatus Phytoplasma australasiaticum’ in antirrhinum, chrysanthemum, china aster, dianthus, amaranthus, french bean, and sunhemp. In-silico restriction fragment length polymorphism (RFLP) analysis indicated that phytoplasma strains infecting Antirrhinum, China aster, Dianthus, French bean, and Sunhemp represented distinct variants within the 16SrII phytoplasma group, with a threshold similarity coefficient of less than 0.98. A loop-mediated isothermal amplification (LAMP) assay was developed for detecting phytoplasma strains infecting the four ornamentals and other studied crops, targeting the phytoplasma 16S rRNA gene sequence with detection in 60 min. The comparative testing of LAMP products using various nucleic acid dyes and a colorimetric dye (hydroxy naphthol blue) confirmed the presence of phytoplasmas in symptomatic samples. This LAMP assay offers superior ease of use, cost-effectiveness, and rapid results for detecting phytoplasmas in symptomatic plants.

The &lt;i&gt;LGALS1&lt;/i&gt; gene polymorphism is not associated with galectin-1 levels in tumor tissue and blood of colon cancer patients

Background: Galectin-1 plays an important role in the pathogenesis of colorectal cancer (CRC). The blood and tumoral levels of galectin-1 could be dependent on the polymorphism of the promotor region of LGALS1 gene. Aim: To analyze an association between galectin-1 levels in tumor tissue and plasma and the genotype of the rs4820293 and rs4820294 polymorphisms of the LGALS1 gene in CRC patients. Materials and methods: The study included a total of 70 inpatients with pathologically verified CRC (International Classification of Diseases 10th Revision codes C18-C20, 39 men and 31 women, mean age 65.4 ± 5.7 years), who were receiving treatment in the Tomsk Regional Oncology Center and Cancer Research Institute of the Tomsk National Research Medical Center from 2020 to 2022. The control group consisted of 70 healthy volunteers (34 men and 36 women, mean age 62.3 ± 7.2 years). Venous blood samples were taken from all study participants and tumor tissue samples were obtained from the CRC patients. Galectin-1 expression in the tumor tissue was assessed by immunohistochemistry and plasma galectin-1 levels by enzyme-linked immunosorbent assay. The LGALS1 gene polymorphisms rs4820293 and rs4820294 were identified by restriction fragment length polymorphism analysis. Results: The distributions of genotype and allele frequencies of polymorphic variants rs4820293 and rs4820294 of the LGALS1 gene in the CRC patients and in the healthy donors were comparable (p 0.05). Calculation of odds ratios did not confirm any association between LGALS1 gene polymorphisms and CRC. However, the rs4820294 polymorphism had a strong association with regional metastasis and tumor differentiation grade (Cramer's V above 0.4, p 0.001). The plasma galectin-1 levels in the CRC patients with the AA genotype of the rs4820294 polymorphism were higher than in the healthy carriers (17.42 versus 12.92 ng/ml, p = 0.040). However, there were no significant differences in the content of galectin-1+ cells in the tumor and galectin-1 in plasma of the CRC patients depending on the genotype of the LGALS1 gene polymorphisms (p 0.05). Conclusion: The LGALS1 gene polymorphism is not associated with CRC, but in the carriers of the rs4820294 variant is related to clinical and morphological parameters of the tumor process. The intratumoral expression and blood levels of galectin-1 in CRC patients are not dependent on the genotype of rs4820293 and rs4820294 polymorphisms of the LGALS1 gene.

Natural Variability of Genomic Sequences of Mal d 1 Allergen in Apples as Revealed by Restriction Profiles and Homolog Polymorphism

Apples are a popular fruit worldwide, with many health and nutritional benefits. However, this fruit is also among those that, particularly in Central and Northern Europe, are allergenic due to the Mal d 1 allergen. Mal d 1 is a homologous allergen to Bet v 1—the main pollen allergen of birch. In this study, two different approaches were used to identify the natural length polymorphism of Bet v 1 homologs in apple varieties, with the aim of characterizing their effectiveness. BBAP (Bet v 1 based amplified polymorphism) and RFLP (restriction fragments length polymorphism) profiles were characterized and compared. RFLP analysis recognizes the genetic diversity of M. domestica Mal d 1 sequences at a relatively low level. In BBAP profiles, the genetic dissimilarity was up to 50%, which appears suitable for intraspecific fingerprinting and serves as an additional method for RFLP analysis. RFLP analysis was able to distinguish some varieties that BBAP could not, such as Sonet.