Polymerase Chain Amplification Research Articles

Old Foe, New Host: Epidemiology, Genetic Diversity, and Pathogenic Characterization of Maize Streak Virus in Rice Fields from Burkina Faso

Rice is of critical significance regarding food security worldwide, including in Africa. Only two viruses impacting rice production in Africa have been deeply investigated for decades: the rice yellow mottle virus ( Solemoviridae) and the rice stripe necrosis virus ( Benyviridae). Using viral metagenomics, we aimed at broadening knowledge on interacting communities associated with plants in rice landscapes and exploring the diversity and epidemiological status of viruses circulating in rice fields from Burkina Faso. We performed an epidemiological survey in this country between 2016 and 2019 involving 57 small farmers’ rice fields under two production systems: rainfed lowlands and irrigated areas. More than 2,700 rice samples were collected without regard to disease symptoms following a regular scheme. Wild and cultivated Poaceae (maize and sugarcane) growing in nearby rice fields were also collected. Unexpectedly, metagenomics detected maize streak virus (MSV) ( Geminiviridae) in analyzed rice samples. Further molecular analyses using rolling circle amplification-polymerase chain reaction showed that MSV was widely distributed and highly prevalent in both rainfed lowlands and irrigated rice areas. MSV-A and MSV-G strains were identified. MSV-G, exclusively identified thus far in wild grasses, was the most prevalent strain, whereas MSV-A, known to cause severe symptoms in maize, was sporadically identified. Using infectious clones in experimental conditions, we confirmed the pathogenicity of both MSV strains in rice. Thus, in addition to contributing to the epidemiological surveillance of rice production in Africa, our results illuminate new epidemiological and pathogenic aspects of one of the most studied plant viruses with significant economic consequences in Africa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

A novel method of multiplex SNP genotyping assay through variable fragment length allele-specific polymerase chain reaction: Multiplex VFLASP-ARMS

Variable Fragment Length Allele-Specific Polymerase Chain Reaction (VFLASP) and Amplification Refractory Mutation System (ARMS) are reliable methods for detecting allelic variations resulting from single base changes within the genome. Due to their widespread application, allele variations caused by Single Nucleotide Polymorphisms (SNPs) can be readily detected using allele-specific primers. In the context of the current study, VFLASP was combined with ARMS method as a novel strategy to enhance the efficacy of both techniques. Clinically important base variations within SNP regions used in the study were detected by a fragment analysis method. To validate the accuracy of the developed VFLASP-ARMS method, specifically designed synthetic sequences were tested using a capillary electrophoresis system. Allele-specific primers exhibit differences solely at the 3′ end based on the sequence of the SNP. Additionally, to increase the specificity of the primers, a base was intentionally added for incompatibility. Therefore, allele discrimination on fragment analysis has been made possible through the 3–6 bp differences in the amplicons.With the optimization of the system, designed synthetic sequences provided reliable and reproducible results in wild-type, heterozygous, and homozygous genotypes using the VFLASP-ARMS method. Hence, our results demonstrated that VFLASP-ARMS method, offers a novel design methodology that can be included in the content of SNP genotyping assays.

Pharmacogenetics: Influence of CYP2C9*2 and *3 alleles polymorphisms on Iraqi type 2 diabetic patients

Abstract Background: Sulfonylureas (SUs) are the most prescribed anti-diabetic drugs. The enzyme responsible for metabolizing of SUs is hepatic cytochrome P4502C9 (CYP2C9). The CYP2C9 gene has numerous allelic variations; among those, the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. The pharmacokinetics of SUs are dramatically impacted by CYP2C9 genotype. Objective: Evaluation of the association of genetic polymorphisms in CYP2C9 gene with the efficacy of glibenclamide (GB), second-generation SUs, by investigating two CYP2C9 allelic variants. Materials and Methods: Blood samples were collected from 113 type 2 diabetes patients. Allele specific amplification-polymerase chain reaction was used to genotype the CYP2C9 gene. Fasting serum glucose, fasting insulin, and glycated hemoglobin (HbA1c) levels were measured as part of the biochemical analysis. Results: The CYP2C9 gene variants were analyzed in a study group. The results exposed that 75 patients carried the wild (CYP2C9*1/*1) genotype, 25 were heterozygote allele (CYP2C9*1/*2) for CYP2C9*2 gene, 4 were homozygous for the variant CYP2C9*2 allele (CYP2C9*2/*2), and 9 were heterozygous for the variant CYP2C9*3 allele (CYP2C9*1/*3). Statistically significant difference was found in mean HbA1c between the mutant and wild alleles group (P = 0.044). The mean HbA1c for those carrying the CYP2C9*2 and*3 alleles (n = 38) was 8.4750 compared to 9.3177 for those carrying the CYP2C9*1 allele (n = 75), which indicate better glycemic control. Conclusion: The accordance of CYP2C9*2 and*3 was found to be associated with severe hypoglycemia (odd ratio [OR] = 2.045). The OR suggests a strong association between CYP2C9*2 and*3 alleles and hypoglycemia. Our findings imply that the diabetic patients with CYP2C9 polymorphism are more likely to suffer hypoglycemia than those with wild type alleles when treated with GB.

Peripheral substitution effects on unnatural base pairs: A case of brominated TPT3 to enhance replication fidelity

The high fidelity poses a central role in developing unnatural base pairs (UBPs), which means the high pairing capacity of unnatural bases with their partners, and low mispairing with all the natural bases. Different strategies have been used to develop higher-fidelity UBPs, including optimizing hydrophobic interaction forces between UBPs. Variant substituent groups are allowed to fine tune the hydrophobic forces of different UBPs’ candidates. However, the modifications on the skeleton of TPT3 base are rare and the replication fidelity of TPT3-NaM remains hardly to improve so far. In this paper, we reasoned that modifying and/or expanding the aromatic surface by Bromo-substituents to slightly increase hydrophobicity of TPT3 might offer a way to increase the fidelity of this pair. Based on the hypothesis, we synthesized the bromine substituted TPT3, 2-bromo-TPT3 and 2, 4-dibromo-TPT3 as the new TPT3 analogs. While the enzyme reaction kinetic experiments showed that d2-bromo-TPT3-dNaM pair and d2, 4-dibromo-TPT3TP-dNaM pair had slightly less efficient incorporation and extension rates than that of dTPT3-dNaM pair, the assays did reveal that the mispairing of 2-bromo-TPT3 and 2, 4-dibromo-TPT3 with all the natural bases could dramatically decrease in contrast to TPT3. Their lower mispairing capacity promoted us to run polymerase chain amplification reactions, and a higher fidelity of d2-bromo-TPT3-dNaM pair could be obtained with 99.72 ± 0.01% of the in vitro replication fidelity than that of dTPT3-dNaM pair, 99.52 ± 0.09%. In addition, d2-bromo-TPT3-dNaM can also be effectively copied in E. coli cells, which showed the same replication fidelity as that of dTPT3-dNaM in the specific sequence, but a higher fidelity in the random sequence context.

Polymorphisms of genes of the renin-angiotensin system: significance in the progression of chronic kidney disease in children

Introduction. A comprehensive analysis of the polymorphisms of the genes of the renin-angiotensin system in children with different nosological forms of nephropathies is a necessary step in determining the clinical and genetic features of the formation of chronic kidney disease (CKD). 
 Aim: to establish the features of ACE (D/I), GT (Thr174Met), AGT (Met235Thr) and AGTR1 (A1166C) gene polymorphisms in CKD children and determine their significance in the progression of the disease. 
 Materials and methods. A retrospective and prospective study included one hundered 1 to 17 years children with nephropathies, examined in the children’s nephrology department of the Samara Regional Hospital over 10 years. In children, the identification of single nucleotide polymorphisms of genes was carried out using an allele-specific polymerase chain amplification reaction using test systems. Evaluation of clinical and paraclinical markers of progression in CKD was carried out twice a year. The results of the study were evaluated with the calculation of the Student–Fisher criteria and correlation analysis.
 Results. in patients with kidney diseases, there was a trend towards an increase in the occurrence of single nucleotide polymorphisms of genes that affect the renin-angiotensin system (RAS). CKD patients at the stage 3–5 accounted for 35%. They had D/D ACE alleles combined with alleles AGT Thr174Met (27.9 ± 6.83%) and Met235Thr (41.86 ± 7.5%), Thr235Thr (30.2 ± 7.0%) and AGTR1 A1166C (37.2 ± 7.32%) more often than in milder CKD (0 and 7.5 ± 3.37%; 5.2 ± 2.94% and 5.2 ± 2.94%; respectively, p ≤ 0.010). 
 Соnclusion. The study of clinical and genetic features of CKD is relevant for the purpose of timely implementation of preventive measures.

Heavy Ion-Responsive lncRNA EBLN3P Functions in the Radiosensitization of Non-Small Cell Lung Cancer Cells Mediated by TNPO1.

In recent decades, the rapid development of radiotherapy has dramatically increased the cure rate of malignant tumors. Heavy-ion radiotherapy, which is characterized by the "Bragg Peak" because of its excellent physical properties, induces extensive unrepairable DNA damage in tumor tissues, while normal tissues in the path of ion beams suffer less damage. However, there are few prognostic molecular biomarkers that can be used to assess the efficacy of heavy ion radiotherapy. In this study, we focus on non-small cell lung cancer (NSCLC) radiotherapy and use RNA sequencing and bioinformatic analysis to investigate the gene expression profiles of A549 cells exposed to X-ray or carbon ion irradiation to screen the key genes involved in the stronger tumor-killing effect induced by carbon ions. The potential ceRNA network was predicted and verified by polymerase chain amplification, western blotting analysis, colony formation assay, and apoptosis assay. The results of the experiments indicated that lncRNA EBLN3P plays a critical role in inhibiting carbon ion-induced cell proliferation and inducing apoptosis of NSCLC cells. These functions were achieved by the EBLN3P/miR-144-3p/TNPO1 (transportin-1) ceRNA network. In summary, the lncRNA EBLN3P functions as a ceRNA to mediate lung cancer inhibition induced by carbon ion irradiation by sponging miR-144-3p to regulate TNPO1 expression, indicating that EBLN3P may be a promising target for increasing the treatment efficacy of conventional radiotherapy for NSCLC.

Assessment of Haptoglobin 2-2 Genotype in Type 2 Diabetes and Cardiovascular Patients in North Indian Population: A Case-control Study

Introduction: Haptoglobin (Hp), an acute phase protein, is a plasma inflammatory marker. It is important in both infectious and non infectious illnesses. When intravascular haemolysis occurs, the dead red blood cells are discharged into the circulation and link to Hp to form the haemoglobin (Hb)-haptoglobin (Hb-Hp) complex that is recognised by the macrophage scavenger receptor CD163. The Hp1 and Hp2 are its two alleles. The Hp2 is formed by a partial duplication of the Hp1 gene. The Hp1 has five exons, but Hp2 has seven exons owing to the duplication of Hp1 third and fourth exon. There are three genotypes of Hp: Hp1-1, Hp2-1, and Hp2-2. The encoded products of genotypes are attached to haemoglobin with varying degrees of affinity: Hp1-1 has the most affinity, Hp2-1 has medium affinity, and Hp2-2 has the lowest affinity. Hp allelic and genotypic variation differs amongst populations and different geographic regions of the world. Aim: To study the haptoglobin 2-2 genotype in type 2 diabetes and cardiovascular patients in north Indian population. Materials and Methods: It was a case-control study and a total of 200 participants were recruited randomly including 50 participants of type 2 Diabetes Mellitus (DM), 50 participants of Cardiovascular Disorders (CVD), 50 participants with Cardiovascular Disorders With type 2 Diabetes Mellitus (CVDWDM) and 50 normal healthy controls from Punjab Institute of Medical Sciences (PIMS) Hospital and clinics and Aashirwad laboratory of Jalandhar, Punjab, India in February 2021 to January 2022. Anthropometric variables were determined by using standard methods and biochemical parameters were measured by commercial available kits. Allele specific Polymerase Chain Reaction (PCR) or Amplification Refractory Mutation System (ARMS) PCR was used to detect Hp genotypes. Data were represented as mean±Standard Deviation and the statistical difference i.e. p<0.05 between all parameters was determined by using the Mann-Whitney U test. Results: Anthropometric parameters i.e. Body Mass Index (BMI) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.023, p=0.01 and p=0.014. Waist Circumference (WC) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.02, p=0.013 and p=0.012 and Hip Circumference (HC) was found to be statistically significant in CVDWDM as compared to CVD, N and T2DM i.e. p=0.02, p=0.017 and p=0.014. However, Apolipoprotein A1 mean value was found to be higher and statistically significant i.e. p=0.03 in Type 2 Diabetes Mellitus (T2DM) as compared to healthy participants. Triglycerides mean value was increased in T2DM as compared to CVD, CVD as compared to N and CVDWDM as compared to healthy controls (p-value=0.012,0.04,0.015, respectively). The findings had shown that a 349 bp band was detected in patients with T2DM, CVD and CVDWDM, implying that the Hp2-2 genotypes were observed but not in healthy controls. Conclusion: The study finds that people with DM, CVD, or CVDWDM all have a 349bp band. It means that the genotypes of the Hp2-2 allele were found in patients of diabetes, CVD and diabetics with CVD but not in the healthy individuals.

Associations between single-nucleotide polymorphisms of the interleukin-18 gene and breast cancer in Iraqi women.

According to long-term projections, by 2030, the world’s population is predicted to reach 7.5 billion individuals, and there will be roughly 27 million new cancer cases diagnosed. The global burden of breast cancer (BC) is expected to rise. According to the Ministry of Health-Iraqi Cancer Registry, cancer is the second largest cause of death after cardiovascular disease. This study investigated the interleukin-18 (IL18) single-nucleotide polymorphisms (SNPs) –607C/A rs1946518 and –137G/C rs187238 using the sequence-specific amplification-polymerase chain reaction approach. Regarding the position –607C/A, there was a highly significant difference between the observed and expected frequencies in patients and controls (χ2 = 3.16 and χ2 = 16.5), respectively. The AA and CA genotypes were associated with significantly increased BC risk (odds ratio [OR], 3.68; p = 0.004 and OR, 2.83; p = 0.04, respectively). Women with the A allele had a 5.03-fold increased susceptibility to BC. The C allele may be a protective allele against BC (OR, 0.19). Although position –137G/C showed no significant differences in the CC genotype distribution (p = 0.18), the frequency of the CC genotype was significantly higher in patients than in controls. In contrast, patients had a significantly higher frequency of GC genotypes than controls (p = 0.04), which was associated with an increased risk of developing BC (OR, 2.63). The G allele frequency was significantly lower in patients than in controls (55.0% vs. 76.2%, respectively). This SNP may be considered a common genotype in the Iraqi population, with the wild-type G allele having a protective function (OR, 0.19) and the mutant C allele having an environmental effect (OR, 2.63).

Association of TMPRSS2Gene Polymorphisms with COVID-19 Severity and Mortality: a Case-Control Study with Computational Analyses.

Coronavirus disease 2019 (COVID-19) is a severe disease caused by a new variant of beta-coronavirus that first appeared in China. Human genetic factors, including polymorphisms, serve pivotal roles in the high transmission of SARS-CoV-2 and the stubbornly progressing sickness seen in a small but significant percentage of infected people; however, but these factors remain ill-defined. A total of 288 COVID-19 patients and 288 controls were genotyped for TMPRSS2 polymorphisms using both restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) and amplification refractory mutation system (ARMS)-PCR techniques. Different genotypes of TMPRSS2 polymorphisms were compared in terms of disease susceptibility and mortality. The statistical analysis showed that minor alleles of all studied variants statistically increased the risk of COVID-19, except for the rs75603675 C > A variant. The T allele of rs12329760 conferred an increased risk of COVID-19. Moreover, the AG/AC/TT/AG combination of genotypes significantly enhanced the risk of COVID-19 in our population. Different haplotypes of rs17854725/rs75603675/rs12329760/rs4303795 polymorphisms, including GACA, GACG, GATG, GATA, AATA, ACCG, ACTG, ACTA, GCCA, and GCTG, were found to be associated with increased risk of the disease (odds ratio > 1). Regarding the clinical and paraclinical characteristics, a statistically significant difference was found between non-severe and severe forms except for gender, platelet, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and underlying diseases. In addition, case genotypes of TMPRSS2 rs17854725 A > G, rs12329760 C > T, and rs4303795 A > G were significantly different regarding severe and non-severe forms of the disease (P-value < 0.001). Specifically, death was more frequent in carriers of the AG genotype of rs17854725 A > G (P-value = 0.022). Patients who carry the minor alleles of the four studied TMPRSS2 variants were rather vulnerable to COVID-19 infection. Our findings indicated that rs17854725 A > G (AA vs. AG and AA vs. GG), rs12329760 C > T (CC vs. CT and CC vs. TT), and rs4303795 A > G (AA vs. AG) genotypes of TMPRSS2 variations are associated with a more invasive disorder pattern. More studies on larger populations are needed to confirm our results.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12010-022-03885-w.

Meat quality - Genetic background and methods of its analysis

Growing consumer awareness is forcing food producers to supply raw material and products of increasingly high quality and health-promoting properties. Knowledge of the genetic background of quality characteristics is taking on great importance, enabling selection based on molecular markers. The increasing throughput of molecular techniques, in combination with an expanding bioinformatics infrastructure, is leading to continual improvement in understanding of the molecular mechanisms influencing meat quality. This has resulted in the identification of polymorphic nucleotides [single nucleotide polymorphisms (SNPs)] showing a relationship with meat characteristics such as tenderness [polymorphism in the calpain (CAPN) and calpastatin (CAST) genes], marbling [diacylglycerol o-acyltransferase 1 (DGAT1)], colour, pH and water-holding capacity (WHC) [CAST, stearoyl-CoA desaturase (SCD) and others], and fatty acid profile (SCD1). An increasingly wide range of methods is used for analysis, from techniques based on amplification of nucleic acids [polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system-PCR (ARMS-PCR)] through Sanger sequencing to high-throughput next-generation sequencing (NGS) techniques. This paper is a review of the literature on polymorphism of genes determining the quality characteristics of meat and molecular methods used to detect them.

Correlation of Helicobacter pylori virulence genotype & severity of mucosal inflammation in gastric biopsies from two geographically diverse regions in India.

H. pylori-associated gastritis in patients from the high-altitude area of Ladakh showed severe gastritis, mucosal nodularity, atrophy, and cancer in comparison to those from North India. This study served to analyze if differences in the H. pylori virulence genotypes decide the extent of gastric mucosal inflammation. Fifty gastric biopsies each from patients with H. pylori-associated gastritis from Ladakh and a tertiary care center in North India were included. The presence of H. pylori strain was confirmed with Warthin starry stain and polymerase chain amplification of the H. pylori-specific 16S rRNA. The cagA, vacA s1, s2, and m1, m2 alleles, and dupA virulence genotypes were studied in all archival samples, followed by their histological correlations. cagA (P 0.009) and vacAs1 m1 (P 0.009) genes were distinctly more in H. pylori strains colonizing the biopsies of North Indian patients. In contrast, the cagA -ve vacAs2 m2 strains were significantly more in H. pylori strain colonizing the biopsies from Ladakhi patients. dupA genotype was almost similarly present in strains from both regions. Among these, only cagA and dupA virulence genes were associated with severe mucosal neutrophilic activity and deep infiltration of H. pylori strains in North Indian patients. Differences in virulence genotypes of H. pylori in gastric biopsies from North Indian and Ladakhi patients were found not significant in deciding the severity of H. pylori-associated gastritis.

Changes in Methylation Patterns of Tumor Suppressor Genes during Extended Human Embryonic Stem Cell Cultures.

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

The effect of inhibiting exosomes derived from adipose-derived stem cells via the TGF-β1/Smad pathway on the fibrosis of keloid fibroblasts.

The main mechanism of keloid formation is that keloid fibroblasts (KFs) apoptosis is inhibited, leading to excessive proliferation. Transforming growth factor-β1 (TGF-β1) is a key signal molecule in the process of regulating cell fibrosis. This paper discusses the effect of adipose-derived stem cell exosomes (ADSCs-EXO) on the proliferation and apoptosis of KFS and its possible mechanism, in order to provide reference for the clinical intervention of hypertrophic scar. ADSCs were isolated and cultured from human adipose tissue, the supernatant was collected, and the exosomes secreted by ADSCs-EXO were extracted by ultracentrifugation. At the same time, KFs were cultured from human keloid tissue to P3 generation, and then divided into four groups: control group, experimental group A, experimental group B and experimental group C. KFs were then cultured with four concentrations of ADSCs-EXO (0, 1, 10, and 100 µg/mL, respectively). After 24 hours, cells in each group were taken to detect the following: proliferation of cells in each group using the cell counting Kit 8 (CCK-8) method, cell migration ability via the Transwell test, cell apoptosis by flow cytometry, collagen synthesis using the hydroxyproline method, messenger ribonucleic acid (mRNA) expression of fibrosis-related genes in each group by real-time fluorescent polymerase chain amplification, and the expression of fibrosis-related proteins in the cells of each group by western blotting. Compared with the control group, the proliferation rate, migration rate, and collagen synthesis levels in the three experimental groups decreased with the increase of ADSCs-EXO concentration, while the apoptosis rate in the three experimental groups increased with the increase of ADSCs-EXO concentration, and the differences were statistically significant (P<0.05). Also, compared with the control group, the relative mRNA and protein expression of alpha-smooth muscle actin (α-SMA), TGF-β1, and Smad3 in the three groups decreased significantly, while the expression of three kinds of mRNA and protein decreased with the increase of ADSCs-EXO concentration, and the differences were statistically significant (P<0.05). ADSCs-EXO may inhibit the proliferation and migration, and promote the apoptosis of KFs by inhibiting the expression of the TGF-β1/Smad pathway.

Implication of VDR Rs7975232 and FCGR2A Rs1801274 Gene Polymorphisms in the Risk and the Prognosis of autoimmune Thyroid Diseases in the Tunisian Population.

Hashimoto’s thyroiditis (HT) and Graves’ disease (GD) are autoimmune thyroid diseases (AITD) that cause hypothyroidism and hyperthyroidism, respectively. The vitamin D receptor (VDR) and the Fey receptor IIA (FcγRIIA), are implicated in the etiology of AITD. This study was conducted to examine the implication of VDR rs7975232 and FCGR2A rs 1801274 variations in the susceptibility and the prognosis of AITD in the Tunisian population. The rs7975232 and rs1801274 (R131H) polymorphisms were analyzed in 162 controls and 162 AITD patients (106 HT and 56 GD) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification of refractory mutation system-PCR (ARMS-PCR), respectively. No significant difference was demonstrated for the rs7975232 between patients and controls. However, a significant association was shown between the rs1801274 polymorphism and AITD or HT in the dominant (p = 0.03 or p = 0.01), codominant (p = 0.019 or p = 0.026) and allelic (p = 0.011 or p = 0.012) models. The rs7975232 was associated with the absence or the presence of anti-thyroglobulin antibody, with the age of AITD and GD patients during the first diagnosis (p = 0.01 and p = 0.009, respectively) and with a high T4 level at the beginning of HT disease. However, the FCGR2A gene polymorphism was associated with a low T4 level at the beginning of GD disease. In conclusion, this study indicates that only the FCGR2A variation could be related to AITD and HT susceptibility and that VDR and FCGR2A gene variations constitute factors to prognosticate the severity of AITD, HT and GD.

Aptamer-Capped nanoporous anodic alumina for Staphylococcus aureus detection

The development of new detection systems for an accurate and rapid identification of pathogens has become an essential challenge in the biomedical field. Herein a highly selective platform based on aptamer-gated nanomaterials for specific Staphylococcus aureus detection is presented. In the proposed design, a nanoporous anodic alumina (NAA) scaffold is loaded with the fluorescent indicator rhodamine B, while pores entrances are capped by a DNA aptamer which selectively recognizes S. aureus cells in less than 1 h. When S. aureus cells are present, the solid is selectively uncapped, and the dye is released to the medium. This nanodevice allows the detection of bacterial concentrations between 2 and 5 CFU mL−1 (in buffer and blood, respectively) and it has demonstrated excellent behavior in terms of specificity and robustness. A set of 25 different clinical samples are analyzed using this simple procedure obtaining excellent results, which agree with conventional hospital reference techniques for the identification of S. aureus. This new method is sensitive, rapid and low cost, and avoids steps such as polymerase chain amplification reaction, which makes it suitable for use in point-of-care detection systems.

Rapid detection of CITES-listed shark fin species by loop-mediated isothermal amplification assay with potential for field use

Shark fin is a delicacy in many Asian countries. Overexploitation of sharks for shark fin trading has led to a drastic reduction in shark population. To monitor international trade of shark fin products and protect the endangered species from further population decline, we present rapid, user-friendly and sensitive diagnostic loop-mediated isothermal amplification (LAMP) and effective polymerase chain reaction (PCR) assays for all twelve CITES-listed shark species. Species-specific LAMP and PCR primers were designed based on cytochrome oxidase I (COI) and NADH2 regions. Our LAMP and PCR assays have been tested on 291 samples from 93 shark and related species. Target shark species could be differentiated from non-target species within three hours from DNA extraction to LAMP assay. The LAMP assay reported here is a simple and robust solution for on-site detection of CITES-listed shark species with shark fin products.

A lateral flow strip in combination with microfluidic device and polymerase chain amplification for rapid and visual detection of lymphatic filarial infection

Background: Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematode parasites which is transmitted by the bites of infective mosquitoes. The diagnosis of LF traditionally relies on the detection of circulating microfilariae (mf) using Giemsa-stained thick blood smears which has several limitations. Materials and methods: In the present study, we developed a lateral flow (LF) strip to be used in combination with a novel microfluidic device and polymerase chain reaction (PCR) for a rapid and visual detection of lymphatic filariae, Brugia malayi/ Wuchereria bancrofti infection in human blood samples. Results: The assay targets B. malayi HhaI gene and W. bancrofti Ssp gene. Prior to perform the assay, no DNA product clean up step required, thus, it can shorten time and reduce cost. The LF strip can detect as low as 10 pg of DNA product and no cross-reactivity with DNA of other parasites such as Gnathostoma spinigerum, cysticercisis soliumnor with DNA of other filariae i.e. Brugia pahangi, Dirofilaria immitis andD. repens. Conclusions: The developed LF strip shows high sensitivity and specificity, in combination with a novel microfluidic device and PCR, can be used as an alternative tool for the diagnosis of lymphatic filariasis.

The effects of placental long noncoding RNA H19 polymorphisms and promoter methylation on H19 expression in association with preeclampsia susceptibility.

The effect of DNA methylation on gene expression triggered it as a susceptibility factor in various diseases including preeclampsia (PE). The pathogenesis of PE is closely associated with the methylation status and genetic variants of relevant genes. Therefore, the aim of the study was to investigate the possible impacts of the placental DNA methylation and rs3741219, rs217727, and rs2107425 polymorphisms of the H19 gene on the PE susceptibility as well as the its mRNA expression. Moreover, eight haplotypes of three loci in the H19 gene were analyzed. In this case-control study, the placentas of 107 preeclamptic and 113 non-preeclamptic women were collected after delivery. The methylation status was assessed by methylation-specific polymerase chain reaction (PCR). The H19 polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism or amplification refractory mutation system-polymerase chain reaction methods. The quantitative real time PCR was used for mRNA expression assay. The placental H19 rs3741219 and rs2107425 polymorphisms were not associated with PE. However, H19 rs217727CT and TT genotypes might be associated with a 9.2- and 17.7-fold increased risk of PE, respectively. The Trs3741219 Crs217727 Crs2107425 and Trs3741219 Crs217727 Trs2107425 haplotypes were significantly lower, whereas the Trs3741219 Trs217727 Crs2107425 and Crs3741219 Trs217727 Crs2107425 haplotypes were significantly higher in PE women. Promoter but not upstream region hypermethylation of H19 gene could be led to decreased risk of PE (MM vs. UM + UU). No significant difference was observed in the placental mRNA expression between two groups. The H19 expression was significantly higher in women with unmethylated (UU), compared to methylated promoter (MM). The H19 expression was 17- and 15-fold higher in H19-rs2107425 CC and CT genotypes in PE women. In conclusion, the H19 rs2107425 polymorphism was associated with a higher risk of PE and increased H19 mRNA expression. The promoter hypermethylation of H19 gene was associated with a lower risk of PE and decreased H19 mRNA expression.

Microchip electrophoretic detection of bacterial lipopolysaccharide based on aptamer-modified magnetic beads and polymerase chain amplification

Bacterial lipopolysaccharides (LPS) are endotoxins, which can cause fever, inflammation, cell and tissue damage, irreversible septic shock and death in mammals as well as other organisms. Herein, a highly sensitive and selective strategy was proposed for detecting LPS using microchip electrophoresis (MCE) based on aptamer-modified magnetic beads and polymerase chain amplification. In this strategy, the biotinylated aptamer for the LPS (LBA) was hybridized partially with the complementary DNA (cDNA), then the double-stranded DNA obtained was immobilized onto the magnetic beads. In the presence of target LPS, the target-aptamer complex was formed and the cDNA would be freed. After a magnetic separation, the cDNA was released into the supernatant and subsequently triggered the polymerase chain amplification. This process eventually generated a large number of output DNA, which could be quantitatively detected by MCE. With the polymerase chain amplification, this designed protocol provided an ultrasensitive detection of LPS down to the 1.1 × 10−14 g/mL with a linear range of 5 orders of magnitude. The proposed method also exhibited a high specificity toward LPS in the presence of other common interfering substances. Moreover, the assay showed a good practical application for LPS determination in serum samples.

EWSR1-NFATC2 Translocation-associated Sarcoma Clinicopathologic Findings in a Rare Aggressive Primary Bone or Soft Tissue Tumor.

In recent years, a novel small round cell sarcoma harboring EWSR1-NFATC2 translocation with immunomorphologic overlap with Ewing sarcoma (ES), myoepithelial tumors, and extraskeletal myxoid chondrosarcoma has emerged. There has not been a case series devoted to describing its detailed clinicopathologic and immunohistochemical characteristics. Six sarcomas harboring EWSR1-NFATC2 fusion transcripts by reverse transcription polymerase chain reaction and amplification of the fusion gene by fluorescence in situ hybridization were identified. The patients were 5 adult men and 1 adult woman. Three were primary bone tumors of the radius and 3 were primary soft tissue tumors. Most tumors showed monomorphic round to epithelioid cells in anastomosing cords and abundant myxohyaline to collagenous extracellular matrix. Two tumors had large areas of a solid, matrix-poor histomorphology. All tumors stained for CD99 and NKX2.2; while EMA, dot-like cytokeratin, and focal WT-1 and SMA were present in some tumors. All but 1 tumor showed poor histologic and radiologic responses to neoadjuvant ES-specific chemotherapy. Local or distant recurrences happened in 4 cases. EWSR1-NFATC2 sarcoma is a novel translocation-associated sarcoma. It presents as either a primary bone or soft tissue tumor, usually exhibits distinctive histopathologic features, and has predilection for long bones of adult men. It consistently shows recurrent fusion gene amplification readily detectable by EWSR1 breakapart fluorescence in situ hybridization, which serves as a diagnostic surrogate. It has potential for local and distant recurrence and histologic progression, and is resistant to Ewing sarcoma-specific chemotherapy.