Polymerase Chain Reaction-restriction Fragment Length Polymorphism Research Articles

Intraspecifična karakterizacija izolata Vibrio alginolyticus prikupljenih iz sustava akvakulture i morskih biotopa u Tunisu korištenjem PCR-RFLP, ECP i OMPS profiliranja

A total of 54 presumptive Vibrio alginolyticus strains isolated from a variety of Tunisian marine biotopes, such as seawater, sediment samples from some bathing areas, and aquaculture farms, were characterized and identified by several biochemical tests. DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, extracellular products (ECP) and outer membrane protein (OMP) profiling were used to evaluate their usefulness as a tool to investigate the Vibrio alginolyticus diversity within this complex group. Results showed that there is great heterogeneity in the diversity observed via the PCR-RFLP method related to the number of genotypes generated by the two enzymes SduI and FaqI tested. This heterogeneity was observed not only according to the origin (seawater, sediment, fish and bivalve aquaculture farms) but also within the same type of sample. The two other methods, ECP and OMP resulted in 32 and 26 profiles, respectively. The discriminatory index determined in this study highlighted the good ability of the PCR-RFLP method to discriminate similar V. alginolyticus strains.

Fully automated ApoE4 assay in plasma and the estimation of the APOE polymorphism

AbstractBackgroundApolipoprotein E (ApoE) is a major cholesterol carrier that supports lipid transport and injury repair in the brain. APOE polymorphic alleles are the main genetic determinants of Alzheimer disease (AD) risk: individuals carrying the ε4 allele are at increased risk of AD compared with those carrying the more common ε3 allele, whereas the ε2 allele decreases risk. Moreover, it has been reported that the APOE4 haplotype has a dose‐dependent association with an increased risk of amyloid‐related imaging abnormalities (ARIA) in individuals receiving disease‐modifying therapy (DMT) for AD. Therefore, the importance of APOE genotyping or phenotyping and its expanding clinical role in dementia field has been highlighted. Here, we developed fully automated chemiluminescence enzyme‐immunoassay (CLEIA) kits for ApoE4 and pan‐ApoE, and evaluated its diagnostic concordance with the APOE genotyping.MethodApoE4 and pan‐ApoE assays for fully automated platform, LUMIPULSE, are a two‐step sandwich CLEIA with a specific treatment process to extract the antigen from the lipid surface in plasma samples during the 1st immunoreaction. The turnaround time for each reaction is within 30 minutes. Healthy volunteers’ samples were used for the measurement of ApoE4 and pan‐ApoE with CLEIA or the PCR‐Restriction fragment length polymorphism (PCR‐RFLP) analysis for the APOE polymorphism. The study design was approved by the ethics committee at H.U. Group Holdings, Inc in the 2022 fiscal year (22‐012‐01).ResultThe ApoE4 kit specifically detected the ApoE4 concentration in plasma samples, and could distinguish ApoE polymorphism. Moreover, the polymorphism could be classified clearly by the ratio of ApoE4 and Pan‐ApoE amount in plasma.ConclusionThe new CLEIA ApoE4 and pan‐ApoE assay robustly distinguishes among APOE4 carrier status in plasma. Assessing ApoE isoform by CLEIA may help not only clarify ApoE metabolism, but also ARIA risk.

Probiotics Potential of Lactic Acid Bacteria from Horse Milk in Tambora, West Nusa Tenggara, Indonesia

This study explores the probiotic potential of lactic acid bacteria (LAB) in horse milk from Tambora, West Nusa Tenggara by examining its functional and probiotic attributes, including bile salt tolerance, low pH, simulated gastric juice (SGJ), simulated intestinal juice (SIJ), antibacterial activity, as well as bile salt hydrolase (BSH) genes. Genotyping of LAB was performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) analysis and then identified based on the 16S rRNA gene. A total of 25 LAB isolates showed the ability to grow at low pH, tolerant to bile, and survived under SIJ and SGJ conditions. The BSH gene was confirmed in three isolates, namely: SK1-28, SK2-30, and SK2-34. Results of RFLP-PCR analysis showed that the LAB isolates were grouped into three groups based on the number and molecular weight of the differences DNA fragments. The 16S rRNA analysis showed that the first two groups were Lacticaseibacillus rhamnosus, whereas the third group was Lactiplantibacillus plantarum. In addition, all Lacticaseibacillus rhamnosusisolates in group I showed the ability to grow at pH 9.0, but not group II. It can be concluded that Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum can be used as the indigenous probiotic bacteria source from Indonesia.

Exploring the Influence of VDR Genetic Variants TaqI, ApaI, and FokI on COVID-19 Severity and Long-COVID-19 Symptoms.

There is increasing evidence regarding the importance of vitamin D in the prognosis of coronavirus disease 2019 (COVID-19). Genetic variants in the vitamin D receptor (VDR) gene affect the response to vitamin D and have been linked to various diseases. This study investigated the associations of the major VDR genetic variants ApaI, FokI, and TaqI with the severity and long post-infection symptoms of COVID-19. In total, 100 Jordanian patients with confirmed COVID-19 were genotyped for the VDR ApaI, FokI, and TaqI variants using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. COVID-19 severity, the most commonly reported long-COVID-19 symptoms that lasted for >4 weeks from the onset of infection, and other variables were analyzed according to VDR genetic variants. In this study, ApaI and FokI polymorphisms showed no significant associations with COVID-19 severity (p > 0.05). However, a significant association was detected between the TaqI polymorphism and the severity of symptoms after infection with the SARS-CoV-2 virus (p = 0.04). The wild-type TaqI genotype was typically present in patients with mild illness, whereas the heterozygous TaqI genotype was present in asymptomatic patients. With regard to long-COVID-19 symptoms, the VDR heterozygous ApaI and wild-type TaqI genotypes were significantly associated with persistent fatigue and muscle pain after COVID-19 (p ˂ 0.05). Most carriers of the heterozygous ApaI genotype and carriers of the wild-type TaqI genotype reported experiencing fatigue and muscle pain that lasted for more than 1 month after the onset of COVID-19. Furthermore, the TaqI genotype was associated with persistent shortness of breath after COVID-19 (p = 0.003). Shortness of breath was more common among individuals with homozygous TaqI genotype than among individuals with the wild-type or heterozygous TaqI genotype. VDR TaqI is a possible genetic variant related to both COVID-19 severity and long-COVID-19 symptoms among Jordanian individuals. The associations between VDR TaqI polymorphisms and long-COVID-19 symptoms should be investigated in larger and more diverse ethnic populations.

LEP (G2548A-G19A) and ADIPOQ (T45G-G276T) gene polymorphisms are associated with markers for metabolic syndrome

Background and aimsThere is a link between genetics with metabolic balance and adiposity homeostasis on metabolic syndrome (MetS). Polymorphism in adipokine genes such as leptin and adiponectin may play an important role in its development. This study aimed to determine the association of the individual and general components of MetS with genetic alterations in LEP (rs7799039 and rs2167270) and ADIPOQ (rs1501299 and rs2241766) genes in the Mexican population.Methods and resultsThe polymorphisms of the LEP gene rs7799039 and rs2167270, together with rs1501299 and rs2241766 polymorphisms of the ADIPOQ gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on 328 individuals (n = 131 MetS). The rs7799039 under the recessive inheritance model was found to be associated with increased risk of MetS (OR = 2.16, 95% CI = 1.06–4.37), dyslipidemia (OR = 7.97, 95% CI = 2.17–29.36), low HDL (OR = 7.01, 95% CI = 1.65–29.71) and hypertension (OR = 13.02, 95% CI = 1.76–96.44); the heterozygote demonstrate a protective effect on MetS (OR = 0.48, 95% CI = 0.28–0.88) and diabetes (OR = 0.09, 95% CI = 0.02–0.53) under the over the dominant model. Haplotype analysis showed linkage disequilibrium between the SNPs of ADIPOQ rs1501299/rs2241766, and their association as risk factors for low HDL and hypertension.ConclusionThe association of rs7799039 with the presence of MetS, suggests a risk factor for the development of dyslipidemia, as well as its heterozygous as a protective factor for DM. There is a linkage disequilibrium between the SNPs of ADIPOQ.

The ENPP1 K121Q polymorphism is associated with obesity-related parameters in Iranian normoglycemic male subjects

Objectives: The K121Q polymorphism of the Ectoenzyme Nucleotide Pyrophosphate Phosphodiesterase 1 (ENPP1) gene has been variably associated with obesity in several populations. However, this association has not been studied in Iranian subjects. It was hypothesized that the K121Q variant might be associated with obesity and related risk factors in subjects with normal glucose tolerance and normal fasting glucose.
 Methods: The K121Q genotypes were determined by PCR-restriction fragment length polymorphism in 377 non-diabetic subjects.
 Results: In males, the frequency of the Q allele was 20.8% and 15.9% (p=0.269) in obese and non-obese subjects, respectively. The ENPP1 genotype (KQ+QQ) was not associated with systolic and diastolic blood pressure, triglyceride, cholesterol, LDL-C, HDL-C, HOMA-IR, or insulin levels in both genders. Male carriers of the KQ+QQ genotype had significantly higher values for BMI (p=0.079), waist (p=0.01), and WHR (p=0.006) than subjects with the KK genotype. Obesity-related parameters were not significantly different between obese and non-obese female subjects.
 Conclusion: Our results suggest that the ENPP1 121Q allele might be associated with obesity and related parameters only among Iranian normoglycemic male subjects.

Polymorphism and promoter methylation-regulated mRNA expression of IFI6 gene affect meat quality in pigs

This study examined the association, promoter methylation of Interferon alpha-inducible protein 6 (IFI6) gene, and their effects on mRNA expression and meat quality traits in pig. Muscle samples (n=300) were randomly collected from the Longissimus thoracis et lumborum (LTL) to determine meat quality traits including drip loss, thawing loss, cooking loss, meat pH [at 45 minutes (pH45min) and at 24 hours (pH24h) post-mortem], shear force (for tenderness), and lightness (L*) in pigs. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Muscle samples (5 per group) with divergent meat pH (low vs high) were selected for promoter methylation study using bisulfate and pyrosequencing. Muscle samples from animals (5 per group) with divergent phenotypes (low vs high meat pH and low vs high drip loss) were used for mRNA expression study by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that a polymorphism in IFI6 gene (g.370A>G) was significantly (P < 0.01) associated with meat pH24h. Furthermore, four CpG regions were identified in IFI6 promoter, of which DNA methylation in a CG site was higher in low meat pH24h group compared to high meat pH24h group (P < 0.05). The mRNA expression of IFI6 gene was significantly (P < 0.01) downregulated in low meat pH24h group than high meat pH24h group. This result postulated that high DNA methylation at CG site in the promoter region might downregulate the IFI6 gene expression, hence lower the muscle pH that might lead to higher drip loss and lower tenderness. Therefore, porcine IFI6 gene might be a potential candidate gene for meat quality trait in pig. Nevertheless, further research is needed to confirm the role of IFI6 gene in controlling meat quality traits in other commercial populations.

The gene polymorphisms of eNOS and MTHFR modulates the development of preeclampsia in Han population

We explored the effects of the endothelial nitric oxide synthase (eNOS) gene −786 T > C and 894 G > T locus polymorphisms and the methylenetetrahydrofolate reductase (MTHFR) gene 1298 A > C and 677 C > T locus polymorphisms on preeclampsia (PE) in pregnant women in Quanzhou area and provide reliable and stable predictors PE. This study included 160 normal pregnant women (normal control group) and 160 women with preeclampsia (PE group). Polymorphisms in eNOS gene and MTHFR were analyzed by the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) technique. eNOS 894 G > T locus and MTHFR 1298 A > C locus had no significant difference between the two groups. In the PE patients, eNOS −786 T allele (OR: 2.07, p = 0.03) and MTHFR 677 C allele (OR: 1.83, p = 0.04) had significantly lower frequency. The nitric oxide (NO) level in patients with eNOS −786 C C was significantly lower than that in those with −786 TT. The homocysteine (Hcy) level in patients with MTHFR 677 TT was significantly higher than that in those with 677 C C. In conclusion, the frequency of the eNOS −786 C C genotype and MTHFR 677 TT genotype are higher in women with PE, which cause lower NO level and higher Hcy level.

The effect of TRIM5 variants on the susceptibility to HIV-1 infection and disease progression in the Polish population.

Tripartite motif containing 5α protein is a factor contributing to intracellular defense mechanisms against human immunodeficiency virus-1 (HIV-1) infection. The studies of TRIM5 variants effects on the risk of HIV-1 infection and the clinical course of disease provided inconclusive results in different ethnic groups. The aim of this study was to investigate the influence of TRIM5 variants on susceptibility to HIV-1 infection and clinical parameters among Polish HIV-1-infected patients. In our study, we investigated 301 HIV-1-infected patients and 186 age-matched seronegative controls. Seven variants of the TRIM5 gene (rs7127617, rs3824949, rs3740996, rs11601507, rs10838525, rs11038628, and rs28381981) were genotyped using both sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. The frequencies of rs7127617 TT genotype and T allele occurrence were lower in HIV-1-infected subjects compared to controls (0.14 vs. 0.26 for T/T genotype and 0.45 vs. 0.54 for T allele), suggesting their possible protective effect (p=0.005 and p=0.007, respectively). Heterozygosity and presence of the T allele at rs3740996 were enriched in controls compared to HIV-1-infected group (0.19 vs. 0.12 for C/T genotype and 0.11 vs. 0.07 for T allele; p=0.03 and p=0.02, respectively). Moreover, rs3824949 CC genotype carriers had a lower viral load than patients bearing rs3824949 GG/CG genotypes (4.0 vs. 4.6 log copies/mL; p=0.049); however, none of the variants affected CD4+ cell count. In conclusion, our data confirm the role of TRIM5 variants in the HIV-1 transmission and the clinical course of HIV-1 infection. The presence of rs7127617 TT genotype and T allele seems to protect against HIV-1 transmission in examined population.

Correlation of Plasma Adiponectin Levels and Adiponectin Gene Polymorphisms with Idiopathic Atrial Fibrillation

Introduction: Adiponectin is a cellular protein secreted by adipocytes, which is closely related to a variety of diseases, including atrial fibrillation (AF). Idiopathic atrial fibrillation (IAF) is defined as AF without hypertension, diabetes, and other underlying diseases. Genetic polymorphism of adiponectin affects serum adiponectin concentration. However, the association of serum adiponectin concentration and its genetic polymorphism with IAF has not been studied. This study investigated the relationship between serum levels of adiponectin, adiponectin gene polymorphisms, and the risk of developing IAF in a Chinese Han population. Methods: Patients with IAF (n = 172, IAF group) and healthy individuals (n = 150, control group) were consecutively and randomly recruited and fasting peripheral blood samples were collected. All participants were examined for serum adiponectin concentrations and the polymorphisms SNP45T>G (SmaI locus, rs2241766) and SNP276G>T (BsmI locus, rs1501299) of the adiponectin gene were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Related clinical data from the two groups were also collected. Results: Plasma adiponectin levels in the IAF group were significantly lower than those in the control group (9.9 ± 2.6 mg/L vs. 16.1 ± 7.0 mg/L, p = 0.006). There were no significant differences among the three genotypes (wild type, mutant heterozygote, and homozygote) of SNP45T>G or SNP276G>T in the prediction value of IAF. The frequency of the T allele of SNP45 T>G was 70.3% in the IAF group and significantly different from that of the control group (71.3%; p = 0.02). In the case of SNP276G>T, the frequency of the G allele was 68.61% in patients with IAF compared to 73.34% in the control group (p = 0.35). Furthermore, a comparison of the clinical data of individuals in the two groups revealed that the left atrial diameter (LAD) in patients in the IAF group was obviously higher than that in the control group (43.3 ± 6.7 mm vs. 37.9 ± 5.1 mm, respectively; p < 0.001). The left ventricular ejection fraction (LVEF) in the IAF group was obviously reduced than that in the control group (54.7 ± 11.9% vs. 60.2 ± 5.6%, respectively; p < 0.001). Conclusions: Low plasma adiponectin levels were significantly associated with IAF. Hypoadiponectinemia can thus serve as an important factor for the incidence of IAF. The genotypes of SNP45T>G and SNP276G>T in the adiponectin gene may not correlate with the occurrence of IAF. However, our results demonstrate that the T allele of SNP45T>G may be responsible for IAF development in the Chinese Han population.

Molecular diagnosis, phylogenetic analysis, and antifungal susceptibility profiles of Candida species isolated from neutropenic oncological patients

BackgroundNeutropenia is the most important cause of life-threatening invasive fungal infections (IFIs). Here, we studied the frequency and antifungal susceptibility profiles of Candida species that colonized or caused infections among neutropenic patients with solid or hematological malignancies.MethodsA total of 362 clinical samples were collected from 138 patients. After initial isolation using a mix of mycological methods, isolates were screened using chromogenic culture media. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for molecular identification. Positive or suspected cases were confirmed using the reference method of sequencing. Antifungal susceptibility testing for voriconazole and caspofungin was carried out using the microbroth dilution method. An in-silico assay was applied for phylogenetic analysis.ResultsThirty-four Candida strains were isolated. C. albicans (47.06%) and C. glabrata (29.41%) were the most frequent strains. Antifungal treatment reduced the chance of Candida colonization by almost 76% in neutropenic patients (OR: 1.759; 95% CI: 1.349 to 2.390; p value: 0.000). An unusual and non-resistant strain, C. lambica, was reported from the bloodstream of a 56-year-old man with hematologic malignancy (HM). Eight isolates were non-susceptible, and one isolate was resistant to voriconazole. Also, four isolates were non-susceptible to caspofungin.ConclusionWe can conclude that there is a cause-and-effect relationship between neutropenia, HM background, and Candida species separated from neutropenic patients, which can lead to possible infections. Further and repetitive studies are recommended using different molecular methods for better prediction and management of fungal infections in neutropenic patients.

Prevalence of 4a/4b polymorphic variants of the eNOS gene introne in patients with different types of encephalopathies

Questions related to the disruption of vasoregulatory processes as essential factor in neurological pathology require further study. The leading role in the vasoregulatory mechanisms is played by endothelial NO synthase which gene has 15 allelic variants. Recent data indicated a probable association between eNOS gene polymorphism and cerebrovascular diseases. The aim of the present work was to study the prevalence of the 4a/4b introne polymorphism of the eNOS gene in patients with various types of encephalopathies and to evaluate the influence of a particular genotype of the studied gene on the occurrence and/or progression of encephalopathy. A total of 96 patients with encephalopathies of various genesis: chronic traumatic encephalopathy, chronic alcohol-induced encephalopathy, chronic vascular encephalopathy, post-infectious encephalopathy were involved in the study. The patients received inpatient treatment in the neurological department of “Ternopil Regional Clinical Psychoneurological Hospital”. Molecular and genetic differentiation of the studied gene variants was carried out by allele-specific PCR or PCR-restriction fragment length polymorphism analysis by standard operational protocols. Analysis of the frequency distribution of eNOS gene 4a/4b polymorphic variant showed that 4b allele prevailed among patients with all types of encephalopathies carriers­. Relative to practically healthy individuals the difference was found only in patients with chronic vascular encephalopathy (CVE), among whom about 39% were carriers of the 4a allele. The presence of the 4a allele was shown to increased the risk of CVE occurrence and/or progression by 4.5 times. The results obtained suggest the reasonability to include the 4a/b intron polymorphism of the eNOS gene in a genetic panel to monitor patients CVE. Keywords: 4a/4b introne, encephalopathies, endothelial NO-synthase, eNOS gene polymorphism

Efek Terapi Citicoline terhadap Pemulihan Kognitif dan Motorik Pasien Stroke Iskemik Akut dengan Polimorfisme ApoE4 rs429358 dan rs7412

Citicoline has a neuroprotectant effect, especially in mild-to-moderate stroke. The presence of the ε4 allele in the ApoE gene is associated with a severe acute ischemic stroke (AIS) phenotype, in addition to neurodegenerative risk This study aims to determine the effect of citicoline on cognitive and motoric recovery in severe AIS biomolecularly, such as ApoE4 rs429358 and rs7412 polymorphisms. A 64-year-old woman was diagnosed with AIS with a 6-hour onset and with a NIHSS score of 25 and MMSE score of 21. The patient’s AIS was biomolecularly severe as proven by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) ApoE4 which yielded 482 bp at rs429358 (codon 112) and 249 bp 167 bp at rs7412 (codon 158) on electrophoretic visualization. The patient received 3x 500 mg intravenous citicoline for 4 days followed by another 1000 mg of citicoline intravenous during polyclinic control for 5 consecutive days. On the 9th day, the patient experienced improvement in cognitive function with an MMSE score of 29 and superior (4/5) and inferior (5/5) motoric strength. Citicoline is a neuroprotectant agent that is beneficial for the recovery of cognitive and motoric function in biomolecularly severe SIA (ApoE4, C/C, Arg112/Arg158) because it can save the penumbra area from infarction.

Association of LIN28B Gene Polymorphisms (rs221634, rs221635, rs314276, rs9404590, and rs12194974) with Non-Hodgkin Lymphoma Susceptibility and Clinical/Pathological Features.

Lymphoma is a common hematopoietic cancer. It has been proposed that LIN28B gene and its variations may have function in cancer progression and metastasis. Therefore, the purpose of this investigation has been to examine the correlation among LIN28B gene polymorphisms (such as rs221634 A>T, rs221635 T> C, rs314276 C>A, rs9404590 T>G, and rs12194974 G>A) as well as the risk of NHL in an Iranian sample. In the current case-control research, 175 individuals with Non-Hodgkin Lymphoma along with 175 normal controls participated; polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology has been utilized to the genotype samples. Our data demonstrated that rs12194974 and the rs221635 variants have been correlated with higher NHL risk, while rs221634 and rs314276 variants were correlated with lower risk of NHL (P≤0.05). In addition, we detected an association between rs221634 and treatment with R-CHOP. No substantial correlation has discovered among rs9404590 polymorphism and NHL in any inheritance models (P≥0.05). This was the first investigation evaluating the correlation among LIN28B gene polymorphisms as well as the occurrence of NLH. Further studies in different ethnic populations and large-scale sample size are needed to support results.

The Relationship Between PD-1(rs2227981) and PD-L1(rs2890658) Polymorphisms and Urothelial Cell Carcinoma.

Background Urothelial cell carcinoma, which is believed to develop from the urothelium (transitional epithelium), is the most common aggressive tumor and accounts for the ten most prevalent cancers in the world. The risk factors for urothelial cell carcinoma are aging, smoking, gender, and genetic alternations. Programmed cell death1 (PD-1) has been widely described as a negative regulator of T-cells by sending inhibitory signals to the T-cell. Through PD-1 binding with PD-L1 (ligand for PD-1), an inhibitory signal is propagated to the T cell. The polymorphisms of PD-1 and PD-L1 lead to an efficient T-cell response and affect an anti-tumor reaction. The polymorphisms of PD-1 and PD-L1 could also affect the carcinogenesis of human cancer, including urothelial cell carcinoma. Therefore, in this study, we evaluated the relation between PD-1(rs2227981) and PD-L1(rs2890658) polymorphisms and the carcinogenesis of urothelial cell carcinoma. Materials and methods This study was conducted using 211 healthy controls and 256 cases of urothelial cell carcinoma among the Japanese population. The DNA samples were extracted from the peripheral white blood cells of each subject. The genotype was detected by using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method. Results C/T (Adjusted OR 1.55, 95% CI:1.02-2.35) and C/T+T/T (OR 1.46, 95% CI:1.01-2.12) genotypes of PD-1 rs2227981 were significant and risk factors for urothelial cancer.Male with A/A genotype in PD-L1 and CT genotype in PD-1 has a significant higher risk factor compared with other genotypes (Adjusted OR 1.83, 95% CI:1.05-3.21). Conclusions and discussion We found that C/T(PD-1) and "A/A (PD-L1) and C/T(PD-1)" were predominant in urothelial cell carcinoma cases. This indicates that C/T(PD-1) and "A/A (PD-L1) and C/T(PD-1)" genotypes could increase susceptibility to urothelial cell carcinoma. However, since our findings indicated that the effects of PD-1 and PD-L1 polymorphisms included discrepancies, additional research will be needed to evaluate the relationship between human cancer and PD-1 and PD-L1 polymorphisms. This is the first study that seeks to find the relation between PD-1(rs2227981) and PD-L1(rs2890658) polymorphisms concerning urothelial cell carcinoma among the Japanese population.

Circulating miRNAs and their functional genetic variants in pseudoexfoliative glaucoma: potential of miR-146a-5p as a diagnostic biomarker.

The etiology and pathogenesis of pseudoexfoliation syndrome (PEX) and its advancement into pseudoexfoliative glaucoma (PEG) are not fully understood. In this study, we aimed to evaluate the possible role played by two circulating microRNAs (miR-146a-5p and miR-196a-5p) in plasma and their functional genetic variants MIR146A rs2910164 and MIR196A2 rs11614913 in susceptibility to PEG or PEX. Plasma miRNA relative expression of 27 patients with PEG, 25 patients with PEX and 27 controls was determined using quantitative RT-PCR, and fold change was calculated using the 2-ΔΔCt method. Genotyping of 300 patients with PEG, 300 patients with PEX, and 300 controls was performed using a PCR-restriction fragment length polymorphism analysis. Plasma miR-146a-5p relative expression was significantly elevated in patients with PEG (3.9-fold) (P < .000) and patients with PEX (2.7-fold) relative to controls (P = .001). The diagnostic ability of plasma miR-146a-5p expression fold change was good for discriminating PEG vs. controls (AUC = 0.897, P < .000), and the optimal decision threshold was 1.83 (sensitivity = 74%, specificity = 93%). Plasma miR-196a-5p relative expression did not differ significantly between study groups. No significant difference in terms of the minor allele frequency or the distribution of genotypes for MIR146A rs2910164 G/C or MIR196A2 rs11614913 C/T was observed between study groups. Circulating miR-146a-5p can contribute to the risk of PEX/PEG. Therefore, we propose that plasma miR-146a-5p can be developed as a potential biomarker for the minimally invasive diagnoses of PEX/PEG and as a potential therapeutic target with further studies.

The Phylogenetic Characterization of Cockroaches (Insecta: Blattaria) in Türkiye and Determination of Their Vector Potential for Medically Important Parasites

This study was performed to investigate the phylogenetic characters of the cockroaches in XXX region for mitochondrial cytochrome c oxidase subunit I (mt-COI), mt-COII, and internal transcribed spacer-2 (ITS-2) gene regions, and to determine their mechanical transmission of medically important parasites. PCR-restriction fragment length polymorphism (RFLP) was performed using mt-COI, mt-COII, and ITS-2 DNA gene regions to identify cockroach species (n=220) collected from different regions. Differentiation of cockroach species was based on RFLP models using two restriction enzymes: AvaI and EciI. For phylogenetic analysis, mt-COI, mt-COII, and ITS-2 DNA barcode regions were amplified with standard primers. The amplicons obtained were purified and sequenced with PCR primers. According to PCR-RFLP, cockroach species were identified as Blattella germanica (n =105), Blatta orientalis (n = 86), and Periplaneta americana (n = 29). A total of 13 haplotypes were detected and Maximum likelihood (ML) analyses revealed that the sequences of all three species for the three gene regions showed monophyletic structuring. The cockroaches were examined for the presence of parasites. It was determined that of the 58 parasitic forms identified belonged to helminth species 46 (79.3%) and protozoan species and 12 (20.7%). The results showed that B. germanica (58.6%) had the highest prevalence, followed by Bl. orientalis (32.8%) and P. americana (8.6%). The study findings not only contribute to the molecular epidemiology of cockroaches but also confirm that they play an important role as mechanical vectors of protozoan and helminth parasites.

Association between GABRG2 Gene Single Nucleotide Polymorphisms and Susceptibility to Ischemic Stroke in a Chinese Population.

Current evidence suggests that Gamma-aminobutyric acid (GABA) receptors are associated with the occurrence and progression of cerebrovascular diseases. The present study investigated the association between single nucleotide polymorphisms (SNPs) in the Gamma-aminobutyric acid type A receptor gamma2 subunit (GABRG2) gene and ischemic stroke (IS). A total of 120 healthy volunteers and 187 patients with IS were recruited. Patients underwent complete neurological assessment and classification with the National Institute of Health Stroke Scale (NIHSS) and the Trial of ORG 10172 in Acute Stroke Treatment (TOAST). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyze SNP sites in 4 different regions (rs211037, rs418210, rs211035, and rs424740) of the GABRG2 gene. SHEsis online platform was used to assess SNP allele and genotype frequencies. Multivariate logistic regression analysis was performed to identify the risk factors for IS. Univariate analysis showed that the T allele and TT genotype distribution for rs211037 were significantly more frequent in the IS group compared to controls (pallele = 0.01, odds ratio (OR) = 1.673, 95% confidence intervals (CI), 1.119-2.500, pgenotype = 0.03). Furthermore, multivariate logistic regression analysis revealed the TT genotype for rs211037 was an independent risk factor for IS (p = 0.017, OR = 1.925, 95% CI, 1.122-3.303). Age was also found to be an independent risk factor, and the older the age, the higher the risk of IS (p = 0.001, OR = 1.047, 95% CI, 1.020-1.073). Finally, subgroup analysis revealed that patients with the rs211037 TT genotype were associated with a higher NIHSS score (p = 0.03), and that large-artery atherosclerosis (LAA) subtype was predominant in patients with the rs211037 TT genotype (p = 0.042). These findings suggest the rs211037 polymorphism in the GABRG2 gene is an independent risk factor for IS in the Chinese population. GABRG2 could thus be a potential biomarker to assess the risk of IS.

The Relationship between Complement Components C1R and C5 Gene Polymorphism and the Values of Blood Indices in Suckling Piglets.

The main mechanism of innate immunity is the complement system. Its components include the protein products of the C1R and C5 genes, which are involved in the classical activation pathway as well as the inflammatory and cytolytic immune responses, respectively. The aim of this study was to determine the relationship between PCR-restriction fragment length polymorphism in C1R (726T > C) and C5 (1044A > C) genes, and the values of hematological and biochemical blood indices in suckling crossbred (Polish Large White × Polish Landrace × Duroc × Pietrain) piglets (n = 473), considering their age (younger, 21 ± 3 days, n = 274; older, 35 ± 3 days, n = 199) and health status. The frequencies of the C5 genotypes deviated from the Hardy-Weinberg expectations. Younger piglets, healthy piglets, piglets that deviated from physiological norms and older piglets with the C1R TT genotype all had lower white and red blood cell indices. In piglets with the C5 CC genotype, younger piglets, piglets that deviated from physiological norms and older piglets, a greater number and/or percentage of monocytes were recorded in the blood. Older piglets also showed an increase in the number of leukocytes and granulocytes, along with a tendency for a decrease in the percentage of lymphocytes in their blood. We concluded that a polymorphism in the C1R gene may exhibit a functional association or genetic linkage with other genes involved in the process of erythropoiesis. Furthermore the relationship between the C5 gene polymorphism and the number and/or percentage of monocytes in the blood may modify the body's defense abilities. Piglets with the CC genotype, having an increased number/proportion of these cells in their blood, probably display a weakened immune response to pathogens or a chronic stimulation of the immune system.

Association analysis between the VDR gene variants and type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is recognized as a complex metabolic which has affected the lives of millions of people around the world. Vitamin D receptor (VDR) gene polymorphisms have been suggested to be a vital contributor to the development of T2DM. However, the association between VDR gene polymorphisms and T2DM remains controversial.We have investigated the association between two VDR gene polymorphisms (rs731236 and rs1544410) and T2DM in an Iranian population. A total of 148 T2DM patients and 100 normal controls were recruited in this study. We used polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis to perform genotyping. The results of the present research revealed that the frequency of the rs731236 C allele was significantly higher in T2DM patients than in normal controls (p = 0.044). The CC genotype of rs731236 was connected with an increased risk of T2DM (OR = 2.85, 95% CI = 1.06-7.69, p = 0.039). However, no significant difference in the frequency of the rs1544410 C allele between T2DM patients and normal controls was observed (p = 0.918). Our findings were suggestive of the rs731236 polymorphism of the VDR as a risk factor for developing T2DM in the Iranian population, while rs1544410 polymorphism may not be associated with T2DM susceptibility. Further research is needed to approve these findings in other populations and to clarify the underlying mechanisms involved in such an association. The online version contains supplementary material available at 10.1007/s40200-023-01323-0.