Allele-specific PCR Method Research Articles

Epidermal Growth Factor Receptor (EGFR) and SMAD4 negatively correlated in the progression of gallbladder cancer in Eastern Indian patients

Background and introductionTwo and half percent of the Indian population suffer from gallbladder cancer (GBC). The primary factors that lead GBC are associated with mutation of several protooncogenes such as EGFR, ERBB2, Myc, and CCND1 along with dysregulation of several tumor suppressor genes such as SMAD4 and CDKN2A. Bacterial infection caused by S.typhi and H.pylori are also hypothesized to be potential factors driving GBC.AimsThis study aims to investigate the molecular mechanisms driving the progression of gallbladder adenocarcinoma in Eastern Indian patients. We specifically focussed on analyzing the mutational status of the KRAS gene, examining the amplification of the ERBB2/Her2-neu gene, and evaluating the expression patterns of six dysregulated genes (CCND1, MYC, EGFR, ERBB2/Her2-neu, CDKN2A, SMAD4). Additionally, we assessed the expression status of TGF-beta, the association between bacterial infections (S. Typhi and H. pylori) and GBC, and the impact of single nucleotide polymorphisms in ERBB2/Her2-neu and CCND1 genes within this population.MethodsSixty-seven samples from GBC-diagnosed patients, 26 other unrelated GBC samples for validation cohort, and 68 gallstone tissue samples were collected for this study. Genomic DNA from normal as well as tumor tissues were isolated, exon 2 and exon 3 of KRAS gene were amplified along, DNA sequenced and analyzed. KRAS codon 12 mutation was detected by allele specific PCR (ASPCR) method. Amplification of UreC A (coding for urease subunit α), VacA (coding for Vacuolating cytotoxin A) and CagA genes (coding for cytotoxin-associated gene A) in H.pylori were amplified using PCR. Similarly, FlicC (coding for flagellin gene C) in S.typhi was amplified using PCR. The ERBB2/Her2-neu SNP I655V, and CCND1 SNP A870G were analyzed using PCR followed by RFLP. Expression studies of CCND1, Myc, CDKN2A, ERBB2/Her2-neu, EGFR, and SMAD4 genes were measured in GBC tumor tissues by sybr green quantitative RT PCR.ResultsThe oncogenes (EGFR and ERBB2/Her2-neu) were statistically significantly overexpressed and the tumor suppressor gene (SMAD4) downregulated in our GBC tumor patient samples. The EGFR and SMAD4 genes were negatively correlated (r = -0.01) in GBC patients and the data is statistically significant and validated through IHC technique. A significant downregulation of TGF-beta had also been observed. Lower frequency (i.e. 11.5%) of KRAS mutation in GBC tumor was observed.ConclusionsEGFR and SMAD4 expression were found to be negatively correlated in GBC tissue samples. ERBB2 overexpression/amplification was observed in 30% of the GBC samples. We also found a low percentage of GBC samples to show KRAS codon 12 mutation in Indian GBC patient population, as had been previously documented in pancreatic cancers.

Unveiling the resistance risk and resistance mechanism of florylpicoxamid in Corynespora cassiicola from cucumber

Florylpicoxamid, a QiI fungicide, demonstrates broad-spectrum activity against a wide range of phytopathogenic organisms belonging to the phyla Basidiomycota and Ascomycota. Nevertheless, the potential for resistance and the underlying resistance mechanisms of Corynespora cassiicola against florylpicoxamid are still not fully understood. We determined the baseline sensitivity levels of 101C. cassiicola isolates to florylpicoxamid. The EC50 values varied from 0.01 to 1.18 μg/mL with an average of 0.50 μg/mL. Laboratory-induced fungicide adaptation of nine wild-type isolates generated seven C. cassiicola mutants exhibiting high level of resistance to florylpicoxamid, all originating from a single parental isolate. The mutants maintained their resistance even after undergoing ten successive cultivations on a medium devoid of fungicides. No cross-resistance was detected between florylpicoxamid and pyraclostrobin, fluopyram, prochloraz, or propineb. Five of the resistant mutants showed an improved compound fitness index (CFI) relative to their parental isolate, whereas the remainder displayed either a reduced or comparable CFI. All seven of the resistant mutants displayed an A37V substitution within the CcCytb protein, which was responsible for the resistance to florylpicoxamid, as validated through molecular docking analysis. Furthermore, an allele-specific PCR (AS-PCR) method for detecting the CcCytbA37V mutation was successfully established. In summarize, the findings of this study indicate a moderate risk of C. cassiicola developing resistance to florylpicoxamid, with the A37V substitution in CcCytb playing a key role in this resistance, detectable through the use of specific AS-PCR primers.

First report of V1016I, F1534C and V410L kdr mutations associated with pyrethroid resistance in Aedes aegypti populations from Niamey, Niger.

Ae. aegypti is the vector of important μ arboviruses, including dengue, Zika, chikungunya and yellow fever. Despite not being specifically targeted by insecticide-based control programs in West Africa, resistance to insecticides in Ae. aegypti has been reported in countries within this region. In this study, we investigated the status and mechanisms of Ae. aegypti resistance in Niamey, the capital of Niger. This research aims to provide baseline data necessary for arbovirus outbreak prevention and preparedness in the country. Ovitraps were used to collect Ae. aegypti eggs, which were subsequently hatched in the insectary for bioassay tests. The hatched larvae were then reared to 3-5-day-old adults for WHO tube and CDC bottle bioassays, including synergist tests. The kdr mutations F1534C, V1016I, and V410L were genotyped using allele-specific PCR and TaqMan qPCR methods. Ae. aegypti from Niamey exhibited moderate resistance to pyrethroids but susceptibility to organophosphates and carbamates. The kdr mutations, F1534C, V1016I and V410L were detected with the resistant tri-locus haplotype 1534C+1016L+410L associated with both permethrin and deltamethrin resistance. Whereas the homozygote tri-locus resistant genotype 1534CC+1016LL+410LL was linked only to permethrin resistance. The involvement of oxidase and esterase enzymes in resistance mechanisms was suggested by partial restoration of mosquitoes' susceptibility to pyrethroids in synergist bioassays. This study is the first report of Ae. aegypti resistance to pyrethroid insecticides in Niamey. The resistance is underpinned by target site mutations and potentially involves metabolic enzymes. The observed resistance to pyrethroids coupled with susceptibility to other insecticides, provides data to support evidence-based decision-making for Ae. aegypti control in Niger.

Genotyping Zebrafish Point Mutant by Allele-Specific Blocking PCR.

Genotyping zebrafish carrying wild-type, heterozygous, or homozygous copies of a mutant allele is often required for investigating gene specific functions, and is routinely performed to differentiate point mutants. In this study, we describe a modified allele-specific PCR method using an additional blocking primer to promote target sequence amplification while suppressing sequences with single mismatch. Using the tp53m214k point mutant as an example, we show that wild-type, heterozygous, and homozygous zebrafish can be easily distinguished using this simple PCR method, which could be widely adapted for genotyping zebrafish with point mutations or small nucleotide insertions/deletions.

#1625 Influence of polymorphisms in genes for interleukin-6 and interleukin-10 on dialysis patients survival

Abstract Background and Aims Chronic inflammation is an important non-traditional risk factor for morbidity and mortality in patients with terminal kidney disease. Associated occurrence of malnutrition, inflammation and atherosclerosis was observed in patients on dialyses and marked as MIA syndrome, a vicious circle in which pro inflammatory cytokines play a key role. It is known that there were individual differences in creation of cytokines, and it is assumed that polymorphisms in the promoter regions of cytokine genes can have a key role in the predisposition to chronic inflammation and its consequences. We investigated association between interleukin-6 -174 G>C, interleukin-10 -1082 G>A, inteleukin-10 -819 T>C genotypes with the survival of patients with terminal renal failure. Method In this study, a total of 128 patients were examined, 77 on the regular hemodialysis (HD) and 51 on the peritoneal dialysis (PD) program of the UKCS Nephrology Clinic in Belgrade. Age, gender, BMI, duration of HD/PD, C-reactive protein (CRP), albumin and IL-6 and IL-10 concentration in serum were analyzed. Analysis of IL6 -174G>C and IL10 -819T>C polymorphisms was performed by the allele-specific PCR method, and analysis of the IL10 -1082G>A polymorphism by the PCR/RFLPs method. The patients were followed for 36 months and during that time 37 patients died. Results Univariate analysis showed that significant predictors of mortality were age (OR= 1.16,95%CI 1.061-1.269, p=0.001), albumin concentration (OR= 0.81, 95%CI 0.656-0.999, p=0.049), CRP (OR= 1.06, 95%CI 1003-1.124, p=0.039) and IL-6 (OR= 1.12, 95%CI 1.038-1.216, p=0.004), as well as IL-6176G>C gene polymorphism (OR= 0.82, 95%CI 0.097-0.805, p=0.018), while in multivariate logistic model separated only the concentration of IL-6 and the polymorphism in the gene for IL-6 -174 G>C. Estimated arithmetic mean of survival in subjects with the IL-6 -174GG genotype was 33.1 months (95%CI 30.8-35.3), GC 34.6 months (95% CI 33.6-35.7) and CC 26.1 (95% CI 20.0-32.2). There is statistically significant difference in survival time in relation to IL-6 -174 G>C genotype (chi-square=11.398; p=0.003). Estimated arithmetic mean survival in patients with the IL-10 -1082 GG genotype was 32.6 months (95% CI 30.5-35.2), in GA 31.5 (95% CI 29.2-33.8) and in AA 36.0 (95% CI 35.9-36.1). Exists statistically significant difference in survival time in relation to 1082G>A genotype (chi-square=9.669; p=0.008). Conclusion Monitoring of survival in 128 patients during 36 months showed that significant worse survival was experienced in patients with IL -6 -174 CC genotype, while polymorphisms in IL 10 gene-1082 G>A and -819T>C did not significantly affect survival. In this group of patients, beside IL-6-174 CC genotype, concentrations of IL-6, C reactive protein and albumin were significant predictors of mortality as well as Kt/V.

Development of an allele-specific PCR (AS-PCR) method for identifying high-methyl eugenol-containing purple Tulsi (Ocimum tenuiflorum L.) in market samples.

Ocimum tenuiflorum L. is a highly traded medicinal with several therapeutic values. Green Tulsi and purple Tulsi are two subtypes in O. tenuiflorum and both have the same medicinal properties. Recent reports have revealed that purple Tulsi contains higher quantities of methyl eugenol (ME), which is moderately toxic and potentially carcinogenic. Therefore, we developed an allele-specific PCR (AS-PCR) method to distinguish the green and purple Tulsi. Using the green Tulsi as a reference, 12 single nucleotide polymorphisms (SNPs) and 10 insertions/deletions (InDels) were identified in the chloroplast genome of the purple Tulsi. The C > T SNP at the 1,26,029 position in the ycf1 gene was selected for the development of the AS-PCR method. The primers were designed to amplify 521bp and 291bp fragments specific to green and purple Tulsi, respectively. This AS-PCR method was validated in 10 accessions from each subtype and subsequently verified using Sanger sequencing. Subsequently, 30 Tulsi powder samples collected from the market were subjected to molecular identification by AS-PCR. The results showed that 80% of the samples were purple Tulsi, and only 3.5% were green Tulsi. About 10% of the samples were a mixture of both green and purple Tulsi. Two samples (6.5%) did not contain O. tenuiflorum and were identified as O. gratissimum. The market samples of Tulsi were predominantly derived from purple Tulsi. The AS-PCR method will be helpful for quality control and market surveillance of Tulsi herbal powders.

Pyrethroid resistance status and co-occurrence of V1016G, F1534C and S989P mutations in the Aedes aegypti population from two dengue outbreak counties along the China-Myanmar border

BackgroundOver the past two decades, dengue fever (DF) has emerged as a significant arboviral disease in Yunnan province, China, particularly in the China-Myanmar border area. Aedes aegypti, an invasive mosquito species, plays a crucial role in transmitting the dengue virus to the local population. Insecticide-based vector control has been the primary tool employed to combat DF, but the current susceptibility status of Ae. aegypti to commonly used insecticides is unknown. Assessment of Ae. aegypti resistance to pyrethroid insecticides and an understanding of the underlying mechanisms of this resistance in the China-Myanmar border region is of significant strategic importance for effectively controlling the DF epidemic in the area.MethodsAedes aegypti larvae collected from Ruili and Gengma counties in Yunnan Province were reared to adults in the laboratory and tested for susceptibility to three pyrethroid insecticides (3.20% permethrin, 0.08% lambda-cyhalothrin and 0.20% deltamethrin) by the standard WHO susceptibility bioassay. Genotyping of mutations in the knockdown gene (kdr), namely S989P, V1016G and F1534C, that are responsible for resistance to pyrethroid insecticides was performed using allele-specific PCR methods. A possible association between the observed resistant phenotype and mutations in the voltage-gated sodium channel gene (VGSC) was also studied.ResultsAedes aegypti mosquitoes collected from the two counties and reared in the laboratory were resistant to all of the pyrethroids tested, with the exception of Ae. aegypti from Gengma County, which showed sensitivity to 0.20% deltamethrin. The mortality rate of Ae. aegypti from Ruili county exposed to 3.20% permethrin did not differ significantly from that of Ae. aegypti from Gengma County (χ2 = 0.311, P = 0.577). By contrast, the mortality rate of Ae. aegypti from Ruili County exposed to 0.08% lambda-cyhalothrin and 0.20% deltamethrin, respectively, was significantly different from that of Ae. aegypti from Gengma. There was no significant difference in the observed KDT50 of Ae. aegypti from the two counties to various insecticides. Four mutation types and 12 genotypes were detected at three kdr mutation sites. Based on results from all tested Ae. aegypti, the V1016G mutation was the most prevalent kdr mutation (100% prevalence), followed by the S989P mutation (81.6%) and the F1534C mutation (78.9%). The constituent ratio of VGSC gene mutation types was significantly different in Ae. aegypti mosquitoes from Ruili and those Gengma. The triple mutant S989P + V1016G + F1534C was observed in 274 Ae. aegypti mosquitoes (60.8%), with the most common genotype being SP + GG + FC (31.4%). The prevalence of the F1534C mutation was significantly higher in resistant Ae. aegypti from Ruili (odds ratio [OR] 7.43; 95% confidence interval [CI] 1.71–32.29; P = 0.01) and Gengma (OR 9.29; 95% CI 3.38–25.50; P = 0.00) counties than in susceptible Ae. aegypti when exposed to 3.20% permethrin and 0.08% lambda-cyhalothrin, respectively. No significant association was observed in the triple mutation genotypes with the Ae. aegypti population exposed to 3.20% permethrin and 0.20% deltamethrin resistance (P > 0.05), except for Ae. aegypti from Gengma County when exposed to 0.08% lambda-cyhalothrin (OR 2.86; 95% CI 1.20–6.81; P = 0.02).ConclusionsAedes aegypti from Ruili and Gengma counties have developed resistance to various pyrethroid insecticides. The occurrence of multiple mutant sites in VGSC strongly correlated with the high levels of resistance to pyrethroids in the Ae. aegypti populations, highlighting the need for alternative strategies to manage the spread of resistance. A region-specific control strategy for dengue vectors needs to be implemented in the future based on the status of insecticide resistance and kdr mutations.Graphical

Association between Genetic Variants Linked to Premature Ovarian Insufficiency and Inflammatory Markers: A Cross-Sectional Study.

Premature menopause (PM) is the cessation of ovarian function before age 40. PM women are more likely to have cardiovascular diseases (CVDs), diabetes, and mental disorders. This is the first study that assessed the association of single nucleotide polymorphisms (SNPs) with anti-heat shock protein 27 (Hsp27), High-sensitivity C-reactive protein (hs- CRP), and PM and serum pro-oxidant-antioxidant balance (PAB), as putative risk factors for CVDs. We aimed to explore the association of oxidative stress markers with eight different SNPs shown to be related to premature menopause. In this cross-sectional research, we included 183 healthy women and 117 premature menopausal women. We determined baseline characteristics for all participants and measured serum hs-CRP, anti-HSP-27 antibody titer, and PAB levels using the established methods. Genotyping for eight SNPs was done using the tetra amplification refractory mutation system polymerase chain reaction (Tetra-ARMS PCR) and allele-specific oligonucleotide PCR (ASO-PCR) methods. We found a significant difference between mean serum PAB levels and the genetic variant of rs16991615 (P=0.03). ANCOVA showed a significant effect of the genotypes rs4806660 and rs10183486 on hs-CRP serum levels in the case and control groups, respectively (P=0.04 and P=0.007). ANCOVA also showed an association between rs244715 genotypes and anti-hsp27 serum levels in the case group (P=0.02). There was a significant effect of the genotypes of rs451417 on the serum hs-CRP level in the control group (P=0.03). There was a significant association of the genetic variants related to PM with oxidative stress and inflammatory markers (serum PAB, anti-hsp27 antibody, and hs-CRP). Accordingly, this seems to be an effective approach to predicting susceptible subjects for cardiovascular and mental disorders as well as various cancers.

Molecular biology techniques for assessing the loss of HLA heterozygosity after allogeneic hematopoietic stem cell transplantation in children with acute leukemia

According to several observations, up to a third of post-transplant relapses in childhood acute leukemia are associated with the loss of heterozygosity of the major histocompatibility complex (HLA). Furthermore, the inefficacy of the graft-versus-leukemia reaction, as evidenced by the lack of therapeutic effect from the infusion of donor lymphocytes, indicates the need for timely detection of this marker to change the treatment strategy in the post-transplant period. To detect the loss of HLA heterozygosity, the method using the commercial KMR-HLA system and analysis using next-generation sequencing (NGS), as well as the method based on the analysis of highly polymorphic STR and VNTR markers located in the HLA loci region on the short arm of chromosome 6, are widely used. The primary objective of our study was to compare the informativeness of these approaches in diagnosing HLA heterozygosity loss in children during the post-transplant period. The obtained data on the frequency of detecting HLA heterozygosity loss were comparable to the literature data and constituted 23 % of cases of post-transplant relapse of B-cell acute lymphoblastic leukemia, 33 % of cases of T-cell acute lymphoblastic leukemia, and 23% of cases of acute myeloid leukemia. We also demonstrated that the method based on STR marker analysis has sensitivity comparable to allele-specific PCR and NGS sequencing methods. Meanwhile, preliminary sorting of the blast population increases the sensitivity of STR analysis and can be recommended in routine practice.

Association analysis of MTHFR (rs1801133 and rs1801131) gene polymorphism towards the development of type 2 diabetes mellitus in Dali area population from Yunnan Province, China.

Type 2 diabetes mellitus (T2DM) is a common complex metabolic disorder that exhibits a strong genetic predisposition. 5,10-methylenetetrahydrofolate reductase (MTHFR) regulates folate metabolism, which has been proposed to be associated with T2DM, although the relationship is inconsistent among different geographical areas. This study aimed to investigate the effects of MTHFR C677T (rs1801133) and A1298C (rs1801131) loci polymorphisms on T2DM susceptibility in the population of the Dali area in Yunnan Province, China. This case-control study included 445 patients with T2DM and 272 healthy control individuals from the Dali area of Yunnan Province. Genotyping of the MTHFR gene polymorphisms was performed using the competitive allele-specific PCR (KASP) method. The effects of genetic variations of the MTHFR gene on T2DM risk were evaluated using odds ratios (OR) and 95% confidence intervals. The results of the present study revealed that the TT genotype (OR = 1.750, P = 0.030) and the T allele (OR = 1.252, P=0.047) at the MTHFR C677T locus were considerably associated with the increased odds of developing T2DM. In addition, the CC genotype (OR = 3.132, P=0.032) at the MTHFR A1298C locus also substantially increased the odds of developing T2DM. The T-A haplotype (OR = 1.305, P=0.030) of MTHFR C677T and A1298C exhibited the increased odds of developing T2DM. Biochemical index analyses showed that patients with T2DM who carried the CT or TT genotype of MTHFR C677T expressed substantially higher levels of fasting blood glucose (FBG), homocysteine (Hcy), and tumor necrosis factor-alpha (TNF-α) than those of the CC genotype. Moreover, the FBG and Hcy levels were considerably higher in patients with T2DM who carried the CC or AC genotype of MTHFR A1298C than those of the AA genotype. No obvious association was observed between these MTHFR polymorphisms and cardiovascular risk in T2DM. Our study suggests that the genetic variations of MTHFR C677T and A1298C are significantly associated with T2DM susceptibility in the population of the Dali area of Yunnan Province, China.

GENOTYPING OF CATTLE BY ALLELIC VARIANTS A1 AND A2 OF THE BETA-CASEIN GENE: EMPLOYING DIFFERENT METHODOLOGICAL APPROACHES (AS-PCR AND ACRS-PCR)

This article addresses the comparative analysis of the efficiency of cattle genotyping based on allelic variants A1 and A2 of the beta-casein gene, employing different methodological approaches. The primary methods employed include AS-PCR (AS-PCR 244 bp and AS-PCR 854 bp) and ACRS-PCR (ACRS-PCR DdeI and ACRS-PCR TaqI). Bioinformatics and laboratory diagnostics methods were used for a comparative analysis of genotyping efficiency. The study results unveiled the advantages and disadvantages of each methodological approach employed, it identified the specificity and accuracy of flanking the experimental fragment of the bovine beta-casein gene and underscored the necessity to optimize typing algorithms based on prevailing conditions when utilizing model objects. Based on the results of the research, an effective general typing algorithm was developed using the AS-PCR and ACRS-PCR methods. The allele-specific PCR method is proposed as the primary approach for routine genotyping of cattle, with ACRS-PCR suggested as a tool to verify results in cases of ambiguous findings and for blind typing of samples, among other applications.

Rare but impaired flavin-containing monooxygenase 3 (FMO3) variants reported in a recently updated Japanese mega-databank of genome resources

Genetic variants of human flavin-containing monooxygenase 3 (FMO3) were investigated using an updated Japanese population panel containing 54,000 subjects (the previous panel contained 38,000 subjects). One stop codon mutation and six amino acid-substituted FMO3 variants were newly identified in the updated databank. Of these, two substituted variants (p.Thr329Ala and p.Arg492Trp) were previously identified in compound haplotypes with p.[(Glu158Lys; Glu308Gly)] and were associated with the metabolic disorder trimethylaminuria. Three recombinant FMO3 protein variants (p.Ser137Leu, p.Ala334Val, and p.Ile426Val) expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75–125 %) as wild-type FMO3 (117 min−1); however, the recombinant novel FMO3 variant Phe313Ile showed moderately decreased FMO3 catalytic activity (∼20 % of wild-type). Because of the known deleterious effects of FMO3 C-terminal stop codons, the novel truncated FMO3 Gly184Ter variant was suspected to be inactive. To easily identify the four impaired FMO3 variants (one stop codon mutation and three amino-acid substitutions) in the clinical setting, simple confirmation methods for these FMO3 variants are proposed using polymerase chain reaction/restriction fragment length polymorphism or allele-specific PCR methods. The updated whole-genome sequence data and kinetic analyses revealed that four of the seven single-nucleotide nonsense or missense FMO3 variants had moderately or severely impaired activity toward trimethylamine N-oxygenation.

Neuropeptide Y, a potential marker for lupus, promotes lupus development

ObjectiveRelationship between neuropeptide Y (NPY) serum levels, NPY genetic mutation with systemic lupus erythematosus (SLE) pathogenesis is yet to be clarified, and role of NPY in development of SLE needs elucidation. MethodThis study included 460 SLE patients, 472 non-SLE cases, 500 healthy volunteers. Serum NPY, matrix metalloproteinase-1 (MMP-1) and MMP-8 levels were tested by ELISA. Genotyping 7 NPY single nucleotides polymorphisms (SNPs) (rs5573, rs5574, rs16129, rs16138, rs16140, rs16147, rs16478) was obtained by Kompetitive Allele-Specific PCR (KASP) method. Pristane-induced lupus mice were treated with NPY-Y1 receptor antagonist, and histological analysis, serological changes of the mice were evaluated. ResultsNPY serum concentrations were significantly increased in SLE patients when compared to that in healthy volunteers, non-SLE cases. Rs5573 G allele, rs16129 T allele, rs16147 G allele frequencies were significantly different between SLE cases and healthy controls. Rs5574 TT + TC genotypes were related to levels of IgG, C3, C4 and erythrocyte sedimentation rate, and rs16138 GG + GC genotypes correlated with SLE cases with anti-double-stranded deoxyribonucleic acid antibody (anti-dsDNA) (+). Serum MMP-1, MMP-8 concentrations were higher in SLE patients, and NPY levels were significantly related to MMP-1, MMP-8 levels. After treatment of lupus mice with NPY-Y1 receptor antagonist, damage of liver, spleen and kidney was alleviated, production of autoantibodies (anti-nuclear antibody (ANA), total IgG, anti-dsDNA) and MMP-1, MMP-8 was down-regulated, and differentiation of CD3+, CD8+ T cells, B cells, monocytes, macrophages, T helper 1 (Th1), Th2, Th17 cells was reversed. ConclusionNPY may be a biomarker for lupus, which may promote occurrence and development of lupus.

Entomological longitudinal surveys in two contrasted eco-climatic settings in Cameroon reveal a high malaria transmission from Anopheles funestus associated with GSTe2 metabolic resistance

BackgroundThe impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos’ propensity to transmit Plasmodium spp.MethodsLongitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes.ResultsAn. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1–5.2), p = 0.03; IRR = 2.5, 95% CI (1.2–5.8), p = 0.01 respectively).ConclusionThis study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance.

Distribution of beta-casein genotype in two generations in cows of the Kholmogory breed

One of the most effective ways to produce A2 milk is marker selection using the results of animal genotyping according to beta-casein genotypes, since A2 milk is produced only by cows with the A2A2 genotype. In this study, the inheritance of the beta-casein gene genotype in two generations of cows of the Kholmogory breed in "Agrofirma "Kholmogorskaya" and "Kholmogorskiy plemzavod", Arkhangelsk region was studied. The A1 and A2 alleles of the beta-casein gene were determined by an allele-specific PCR method. As a result of the study, it was found out that the frequency of the A1 and A2 alleles of the beta-casein gene in daughter offspring and mother cows practically did not change in one generation (A1 allele from 53 to 54 % and A2 allele from 46 % to 47 %; A1 allele from 68 to 61 % and A2 allele from 32 to 39 %). This fact indicates the absence of breeding pressure. Since the task of modern animal husbandry is to create herds of the Kholmogory breed with the A2A2 genotype for the production of high-quality milk, then for further breeding, attention should be paid to animals with the A1A2 and A2A2 genotypes. Having modeled the selection in herds, that is, excluding mothers with the A1A1 genotype from reproduction, and calculating the distribution of genotypes of descendants in the remaining part of the maternal stock of both herds, it was found that the proportion of descendants with the A1A1 genotype decreases in farms, the relative number of heterozygous daughters in the breeding farm increased by 5.0 %, and in the agrofirm decreased by 6.3 %, but at the same time, the number of A2A2 daughters increased by 6.7 and 4.0 %, respectively.

Association of STAT3 gene polymorphisms with systemic lupus erythematosus in a Chinese Han population.

Evidence supports the important role of STAT3 in SLE; however, association between STAT3 gene polymorphisms and SLE risk needs discussion. Three hundred SLE patients and 380 healthy controls from Chinese Han population were included. DNA is extracted from peripheral blood mononuclear cells and the clinical characteristics of patients are collected. STAT3 gene polymorphisms (rs6503695, rs744166, rs9912773, and rs12601982) were genotyped by the Kompetitive Allele-Specific PCR (KASP) method. SPSS 26.0 was utilized to analyze the genetic susceptibility of SLE and STAT3 gene polymorphisms. Frequencies of genotypes CT, TT, and TT+CT were significantly lower in SLE patients compared with those in healthy controls with respect to rs6503695 (p = .007, p < .001, p = .001). Frequencies of rs744166 genotypes AG, AA, and AA+AG were decreased in SLE patients as compared to those in healthy controls (p = .034, p = .006, p = .009). The recessive models (CC vs GG+GC) for rs9912773 and (AA vs GG+GA) for rs12601982 were significantly related to SLE patients (p = .014, p = .035). Moreover, allele C of rs6503695 was related to optic nerve damage in SLE patients (p = .036). rs744166 allele G was correlated with positive rash and albuminuria in SLE patients (p = .006, p = .014). For rs9912773, SLE patients carrying genotype GG had higher serum C3 and C4 levels compared to genotype GC+CC (p = .029, p = .028). The rs12601982 allele G was strongly associated with positive hypocomplementemia in SLE patients (p = .034). SLE patients carrying genotypes GG, GC, and CC had different SLEDAI score for rs12601982 (GG vs GC vs CC, p = .003). STAT3 gene polymorphisms associated with SLE susceptibility.

Analysis of polymorphic variants of MSTN, CAST, PRLR genes associations with economically useful qualities of vyatka breed horses

The relationship of polymorphic variants of MSTN, CAST, PRLR genes with working qualities and body types of Vyatka horses was studied. The purpose of the research is to assess the genetic and breeding potential of the Vyatka breed horses, as well as to study the relationship between the MSTN, CAST, PRLR genotypes and the economically useful qualities of horses. The method of DNA extraction from Vyatka horse hair follicles using ExtraGene DNA Prep. was applied. When scanning the mutations in the loci MSTN (n=43), CAST (n=41) and PRLR (n=41), DNA amplification the method of allele-specific PCR was performed. The frequency of alleles and genotypes was calculated using MS Excel 10. As a result of research, the dependence of Vyatka horses working qualities on the frequency of occurrence of myostatin MSTN alleles (g.66493737 T&gt;C) was noted. The horses with a higher occurrence of the MSTN/C allele have more productive movements than the individuals with the T/T genotype typical for aborigines. Vyatka horses with the T/T genotype are more versatile, and also show better results in sledding, while the horses with the T/C genotype are better under saddle. The horses with the T/T genotype have the highest bony index but the lowest massiveness index. The MSTN T/T (0.581) homozygous genotype for the "wild" allele predominates in Vyatka horses. A relation ship between body types and the calpastatin gene (CAST) has been revealed for the first time in horse breeding. The horses with the G/A genotype turned out to be the most massive and bony, the horses with the A/A genotype were lighter, the highest frequency of occurrence of the CAST G/A genotype (0.463) was noted, the CAST G/G genotype is rare in the breed (0.171). A relationship between the frequency of occurrence of prolactin receptor genes (PRLR) and body types of horses was not found. The frequency of occurrence of PRLR C/C (0.366) and PRLR G/C (0.390) genotypes is approximately identical, the PRLR G/G genotype is less common (0.244). The study of genes associated with economically useful qualities in all breeding stallions will enable to conduct more efficient breeding, using the desired genotypes, which is important for small breeds.

Serotipado de Escherichia coli aisladas de pollos de engorde y determinación de resistencia a la Colistina

Systemic infections by avian pathogenic Escherichia coli (APEC) are economically damaging to poultry industries Worldwide. E. coli strains of serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster that controls O antigen synthesis generally varies among different E. coli serotypes. In this study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared, and it was also aimed to search for Colistin resistance on a molecular basis. For the research, 200 swab samples were taken from 200 chickens suspected of colibacillosis in broiler poultry farms located in the vicinity of Aydın, İzmir, and Manisa Provinces in Turkey 2022. Bacterial growth was obtained from 92% of the samples, and microbiological analysis identified 108 (54%) Escherichia coli isolates. In addition, Klebsiella spp. was identified in 35 (17.5%) samples, Proteus spp. in 23 (11.5%), Pseudomonas spp. in 18 (9%), and no bacterial growth was observed in 16 (8%) samples. mcr-1 (309 bp) and mcr–2 (567 bp) genes responsible for Colistin resistance was investigated in plasmid DNA extracted from 108 E. coli isolates obtained in the study, using the PCR method. However, neither mcr-1 nor mcr–2 genes were detected in any of the samples. In conclusion, the allele-specific PCR method was found sensitive and applicable for APEC identification and multiple drug resistance emerged in E. coli strains isolated according to the antibiogram results.

Method Development of Nested Allele-Specific Multiplex Polymerase Chain Reaction (NASM-PCR) for the Irritable Bowel Syndrome (IBS)-related Gene Polymorphisms

Polymorphisms in the genes of G-protein subunit beta 3 (GNB3); rs5443, tryptophan hydroxylase 1 (TPH1); rs211105 and rs4537731, tryptophan hydroxylase 2 (TPH2); rs4570625 and sodium voltage-gated channel alpha subunit 5 (SCN5A); rs1805124, have known to cause the abnormalities in the gastrointestinal tract that are implicated to irritable bowel syndrome (IBS) predisposition. Upfront genetic polymorphism genotyping in IBS-related gene polymorphisms will help to intervene and guide the decision-making in the management of IBS patients. This study aimed to develop a genotyping method to detect the respective polymorphisms using nested allele-specific multiplex polymerase chain reaction (NASM-PCR). A combination of nested and allele-specific multiplex PCR method was developed to determine the five single nucleotide polymorphisms (SNPs) mentioned. Annealing temperature, annealing and extension times, and the concentrations of MgCl2, primers, and DNA samples were optimized in the PCR. Sanger sequencing was performed to validate the genotyping results. NASM-PCR for IBS-related gene polymorphisms were successfully developed. DNA bands which correspond to the genes and SNPs have shown 100% homologous with the gene database. The developed method of NASM-PCR was reproducible and specific to be used for determining the respective polymorphisms of IBS. Notably, the described method can easily be integrated into other laboratories for population study or clinical use.

Comparative analysis of A1 and A2 allele detection efficiency for bovine CSN2 gene by AS-PCR methods.

This study was aimed at conducting a comparative analysis of the efficiency in genotyping cattle by beta-casein locus using the allele-specific PCR methods. The results of the study have demonstrated the necessity to optimize the protocol for the use of AS-PCR to detect alleles A1 and A2. It was found that the use of non-optimized PCR protocols led to the genotyping errors, manifested regarding beta-casein locus (CSN2). The impossibility of using the touchdown PCR as an optimization instrument for AS-PCR was proven. The elaborated typing protocols were used to study the genetic structure of the cattle populations of different breeds, reared in Ukraine - Ukrainian Red-and-White dairy, Ukrainian Black-and-White dairy (two populations), and Charolais. It was found that locus CSN2 was polymorphic in all the cattle populations. The frequencies of allele A2 varied within 0.34-0.91 depending on the population of the animals, which may be conditioned by the specificities in the selection work. No deviation from the Hardy-Weinberg equilibrium was found in any investigated population of cattle.