TaqMan Real-time Polymerase Chain Reaction Research Articles

Association of F11R polymorphisms and gene expression with primary Sjögren's syndrome patients.

F11R gene encodes junctional adhesion molecule-A protein (JAM-A), which is expressed in various types of cells and is involved in leukocyte extravasation during inflammation. Sjögren's syndrome (SS) is a chronic systemic inflammatory disease that involves lymphocytes invasion of exocrine glands. F11R has been studied in autoimmune diseases, but any association between F11R and SS has not yet been investigated. Therefore, experiments were undertaken to examine the relationships among F11R gene polymorphism, messenger RNA (mRNA) expression and SS patients. Three hundred and twenty-nine patients with SS, and 223 healthy controls were enrolled in their recruitment from the Kaohsiung Medical University Hospital. Genomic DNA was extracted from peripheral blood mononuclear cells and gene polymorphisms were genotyped by TaqMan real-time polymerase chain reaction (PCR). F11R mRNA expression was quantitated by quantitative real-time PCR with TaqMan Gene Expression Assay. Our study showed the genotype -688A/C (rs6695707) was not found in relation to SS patients. The odds ratio of -436A/G (rs12567886) genotype was notably associated with less susceptibility of SS in human leukocyte antigen (HLA)-DR2 negative and HLA-DR3 negative individuals. F11R mRNA expression was lower in SS patients than in the cells of healthy controls. The result indicated that G allele of -436A/G genotype has the potential protective effect against SS disease condition. F11R mRNA was expressed significantly lower in SS patients.

Association Between the SLC1A1 Glutamate Transporter Gene and Obsessive-Compulsive Disorder in the Chinese Han Population.

BackgroundObsessive-compulsive disorder (OCD) is a common, serious and genetically related mental illness; the etiology of OCD has not yet reached a definitive conclusion. Multiple evidence suggests that the glutamatergic system plays a major role in the pathophysiology of OCD. However, subsequent studies on the glutamate transporter gene are not consistent. OCD is a heterogeneous disease. To resolve the complex genetic basis of OCD, division the disorder into different subphenotypes is an effective method for studying the pathogenesis of OCD.MethodsWe recruited 438 OCD patients and 465 age- and sex-matched controls from a Chinese Han population. rs10491734, rs3780412, rs301434 and rs3087879 SNPs were genotyped by real-time TaqMan polymerase chain reaction, and the chi-squared test was used to compare allele and genotype frequencies of variants between the two groups.ResultsThe genotype of rs301434 was statistically significant in total patients with OCD and the controls. After grouping by age and gender, the genotype of rs301434 was statistically significant in early-onset OCD, late-onset OCD as well as male OCD, the allele and genotype of rs3780412 was associated with late-onset OCD. Haplotype analysis showed that four loci haplotypes (G-A-A-G and G-G-A-G) were associated with total OCD, (G-G-A-G) was associated with female OCD, (G-A-G-G) was associated with male OCD, (G-A-A-G and G-G-A-G) were associated with late-onset OCD.ConclusionThis study provides suggestive evidence that SLC1A1 may be involved in the development of OCD in the Han population. However, these findings require further replication.

Detecting Heterobasidion irregulare in Minnesota and Assessment of Indigenous Fungi on Pines

Heterobasidion irregulare is one of the most problematic forest pathogens in the northern hemisphere, but has only been found relatively recently in the north central United States. Discovered in Wisconsin in 1993, but probably established sometime before that, it quickly spread throughout the state. In November 2014, it was found in southeastern Minnesota. Field surveys were then conducted throughout Minnesota with the focus in the southeast near the initial discovery. To find additional infection sites, surveys were conducted with accompanying aerial imagery of red pine (Pinus resinosa Aiton) stands that were previously thinned. Samples were collected from selected sites with dead and dying trees as well as samples from stumps in recently thinned pine stands. These samples were processed first with a nested polymerase chain reaction (PCR) protocol, which was replaced by a real-time PCR assay after its development. No samples tested positive for H. irregulare using these methods and no cultures from isolations were obtained outside the original infection area. Other indigenous fungi were also identified. The majority were wood decay fungi in the Basidiomycota. A spore collection study was also conducted after field surveys. Automated rotary arm spore collectors were used and assayed with an ITS TaqMan real-time PCR assay. Collectors were placed strategically in different areas of Minnesota. A positive control was used in an infected red pine plantation in Wisconsin and this location had the highest number of spores trapped, with 63,776 over a week period. Spores of H. irregulare were detected at several sites in Minnesota, with the highest spore total observed in traps at 413 over a week period. All other locations sampled also had some spores collected except Itasca State Park located in northwestern Minnesota. The weekly deposition of spores ranged from 0 to 1.26 m−2 h−1. Low spore levels occurring in Minnesota indicate that some spores are present, but they are currently being detected in amounts that may not be sufficient for colonization to be successful.

The SERPINA4 rs2070777 AA Genotype is Associated with an Increased Risk of Recurrent Miscarriage in a Southern Chinese Population.

BackgroundMany inflammation-related gene polymorphisms are associated with susceptibility to recurrent miscarriage. SERPINA4 is involved in inflammation and is associated with susceptibility to a variety of diseases, but its relevance in recurrent miscarriage is unclear. Therefore, this study aimed to investigate the relationship between SERPINA4 gene polymorphisms and susceptibility to recurrent spontaneous abortion.MethodsTwo SERPINA4 polymorphisms were genotyped in 631 patients with recurrent miscarriage and 771 controls by TaqMan real-time polymerase chain reaction, and the strength of each association was evaluated through 95% confidence intervals (CIs) and odds ratios (ORs).ResultsThe results showed that SERPINA4 rs2070777 AA genotypes were associated with an increased risk of recurrent miscarriage (AA vs AT/TT adjusted OR=1.409, 95% CI=1.032–1.924, P=0.0309), and we also found a significant association between the rs910352 T allele in the SERPINA4 gene and susceptibility to recurrent miscarriage (CT vs CC adjusted OR=1.579, 95% CI=1.252–1.992, P=0.0001; TT vs CC adjusted OR=1.524, 95% CI=1.134–2.049, P=0.0052). The combined analysis of two SNPs of the SERPINA4 gene revealed that carriers with one to two unfavorable genotypes were associated with a higher risk for recurrent miscarriage compared with individuals with no unfavorable genotypes (adjusted OR=1.257, 95% CI=1.019-1.550). Moreover, our study indicates that having one to two unfavorable genotypes is associated with an increased risk of recurrent miscarriage in women 35–40 years of age.ConclusionOur study suggests that SERPINA4 rs2070777AA genotypes might contribute to an increased risk of recurrent miscarriage in a southern Chinese population.

Development of TaqMan Real-Time Polymerase Chain Reaction Assay for Detection of Chicken Anemia Virus in Newcastle Disease Vaccines.

Exogenous chicken anemia virus (CAV) has been detected in commercial poultry vaccines in various countries of the world. The presence of unwanted CAV in vaccines not only influences the epidemiology of chicken infectious anemia disease, but may also lead to vaccine failure and confusing results when vaccine responses are monitored. To detect CAV in contaminated vaccines, nucleic acid testing (unlike conventional testing) has a shorter processing time and does not require cell culture or live animals. The aim of the current study was to develop a TaqMan real-time polymerase chain reaction (PCR) assay to detect and quantify CAV in poultry vaccines and investigate CAV contamination in Razi live Newcastle disease vaccines. The TaqMan real-time PCR assay was set up, optimized, and validated in successive experiments. A standard plasmid pUC-VP2 containing viral protein 2 of CAV was constructed and used in the assay to generate a standard curve to quantify CAV genomes. A clear linear correlation was observed between threshold cycle (Ct) values and plasmid copy numbers in the amplification plots of 10-fold serial dilution of the plasmid. Total DNA of three samples of each of four different Razi live Newcastle disease vaccines, namely LaSota, B1, clone.12IR, and thermo-resistant strains, were extracted and subjected to real-time PCR assay. No CAV contamination was detected in the Razi Live Newcastle vaccines. The developed TaqMan real-time PCR assay provides a quick, specific, and sensitive method for use in detecting CAV in quality control vaccine testing and viral load studies.

Anti-Early Stage of Bacterial Recolonization Effect of Curcuma longa Extract as Photodynamic Adjunctive Treatment.

Objective To evaluate the amount of Fusobacterium nucleatum (F. nucleatum) and Prevotella intermedia (P. intermedia) on subgingival recolonized plaque after mechanical debridement and photodynamic treatment by using blue light-emitting diodes (LEDs) in combination with topical Curcuma longa gel extract. Methods A total of 12 subjects with stage III grade B periodontitis were recruited for the study. Maxillary posterior teeth with periodontal pocket >4 mm were selected. These teeth were examined for periodontal clinical data at baseline and at 1, 2, 4, and 6 weeks after treatment. All remaining teeth were treated by scaling and root planing (SRP). Then, the teeth were bilaterally divided using randomized split-mouth design with and without photodynamic adjunctive therapy (PDT). Samples of the subgingival microbiota were obtained in each visit. All samples were analyzed by multicolor TaqMan real-time polymerase chain reaction (PCR) for the presence of target bacteria. Results Throughout the six-week follow-up, long-term improvement of probing depth and bleeding on probing was revealed on the PDT group. The number of subgingival F. nucleatum and P. intermedia also significantly reduced, compared to the baseline. There was a statistically significant recolonization in F. nucleatum and P. intermedia number after 2 and 4 weeks of conventional SRP, respectively. Our quantitative PCR method showed no significant recolonization of those subgingival bacteria on PDT sites throughout the 6-week study duration. Conclusion The results showed that adjunctive photodynamic treatment by using blue LEDs in combination with topical Curcuma longa gel extract was effective to alter the recolonization patterns of F. nucleatum and P. intermedia after conventional debridement.

Risperidone-Induced Obesity in Children and Adolescents With Autism Spectrum Disorder: Genetic and Clinical Risk Factors.

Aims: Obesity is a significant problem for patients taking atypical antipsychotics. There were two aims of our study. The first aim was to compare the prevalence of overweight and obesity between children and adolescents with autism spectrum disorder (ASD) treated with risperidone with the general pediatric population. The second aim was to investigate the association of the HTR2C -759C>T, ABCB1 1236C>T, ABCB1 2677G>T/A, and ABCB1 3435C>T polymorphisms with risperidone-induced overweight and obesity in children and adolescents with ASD. Methods: Body weight and height were measured in 134 subjects. Overweight and obesity in children and adolescents were classified using the International Obesity Task Force (IOTF) criteria. Genotyping was performed by TaqMan real-time polymerase chain reaction (PCR). Results: Our study found that the prevalence of overweight and obesity was significantly higher in children and adolescents with ASD treated with risperidone compared with healthy individuals (p = 0.01 and p = 0.002). The genetic polymorphisms of HTR2C –759C>T, ABCB1 1236C>T, ABCB1 2677G>T/A, and ABCB1 3435C>T were not associated with overweight/obesity in children and adolescents with ASD treated with risperidone after adjustment for multiple comparisons by the method of Bonferroni. Additionally, haplotype analysis revealed that there was no significant association between ABCB1 3435T-2677T/A-1236T haplotype and overweight/obesity. In multivariate logistic regression, after adjustment by the Bonferroni correction, there was only the duration of risperidone treatment that was significantly associated with overweight/obesity in children and adolescents with ASD. Conclusions: The findings suggest that children and adolescents with ASD treated with risperidone are at a higher risk of obesity, especially patients with extended treatment with risperidone. For the pharmacogenetic factors, –759C>T polymorphism of HTR2C gene and 1236C>T, 2677G>T/A, and 3435C>T polymorphisms of ABCB1 gene were not likely to be associated with the susceptibility to overweight/obesity in children and adolescents treated with risperidone. Due to the small sample size, further studies with a larger independent group are needed to confirm these findings.

Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae).

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/μl to 1 × 10-6 pg/μl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

A Novel Arthropod Host of Brucellosis in the Arid Steppe Ecosystem.

Brucellosis is a severe public health problem in the Inner Mongolia regions of China. The recent prevalence of brucellosis outbreaks may be attributed to an increase in the activity of ticks and other air-borne vectors. Dermacentor nuttalli (D. nuttalli) is a native tick species of Inner Mongolia; similar to other tick species, D. nuttalli carries a variety of pathogens that can be transmitted to a wide range of animals. In this study, we have investigated the potential of D. nuttalli in transmitting brucellosis. From 2015 to 2019, 2,256 ticks were collected from 23 different pastoral areas of Hulun Buir. Brucellosis pathogen was detected using DNA extracted from different developmental stages of ticks. Salivary gland and midgut tissue samples were used as templates to amplify Brucella Bscp31 gene (Brucella genus-specific gene) by using TaqMan Real-Time polymerase chain reaction (PCR). To detect the presence of Bscp31 protein, which is specific to Brucella spp., in the midgut and salivary gland tissues of D. nuttalli, Western blotting and immunofluorescence were performed. Additionally, Brucella spp. were isolated using a culture medium. Tick samples were identified as D. nuttalli. Different percentages of Brucella genus-specific genes could be found in the tick samples. From 2015 to 2019, the positivity rate for the detection of Bscp31 gene in D. nuttalli ranged from 0.00 to 87.80%, with the highest rate of 89.00%. In addition, Brucella genus-specific genes were successfully detected in the samples isolated from all the developmental stages and anatomical regions of ticks. Bscp31 protein was present in the midgut and salivary gland of D. nuttalli. Further, B. melitensis biotype 3 was isolated from eggs and engorged adults of D. nuttalli. These findings demonstrate that D. nuttalli is a potent, long-term carrier of Brucella spp. that can exhibit transovarial transmission potential, presenting D. nuttalli as a novel arthropod host for Brucella spp. This study, therefore, indicates the potential risk of transmission of brucellosis via tick bites among animals as well as human beings.

Association of microRNA 17 host gene variant (rs4284505) with susceptibility and severity of systemic lupus erythematosus.

ObjectiveMicroRNAs are large family clusters of small noncoding RNAs that implicated in genetic and epigenetic regulation of several immunological processes and pathways. As an epigenetic modifier, the microRNA 17‐92 cluster host gene (MIR17HG) has been shown to regulate the expression of genes involved in systemic lupus erythematosus (SLE) pathway. This study aimed to explore the association of MIR17HG (rs4284505; A>G) variant with SLE development and phenotype in a sample of the Eastern Mediterranean population.MethodsA total of 326 participants (163 patients with SLE and 163 healthy controls) were enrolled in this study. The different genotypes of the MIR17HG (rs4284505) variant were characterized using the TaqMan real‐time polymerase chain reaction technique. Association with the available clinical and laboratory data, including the systemic lupus erythematosus disease activity index (SLEDAI), was also executed.ResultsThe MIR17HG (rs4284505) variant showed a protective effect against developing SLE under heterozygote (A/G vs A/A; odds ratio [OR] = 0.10, 95% confidence interval [CI] = 0.05‐0.20, P < 0.001) and dominant (A/G+G/G vs A/A; OR = 0.39, 95% CI = 0.25‐0.61, P < .001) models. This association was consistent even after SLE stratified by lupus nephritis. In contrast, rs4284505 (G/G) genotype conferred increased susceptibility to SLE (G/G vs A/A+A/G; OR = 2.15, 95% CI = 1.31‐3.53, P = .002). Moreover, the rs4284505 variant showed a statistically significant association with mucocutaneous lesions and SLEDAI scores (all P < .05).ConclusionThis study is the first one to explore that the MIR17HG rs4284505 is associated with SLE risk; (A/G) genotype conferred a protective effect, while the (G/G) genotype showed increased susceptibility to SLE and association with the disease severity in the study population.

Clinical Features, Replication Competence, and Innate Immune Responses of Human Adenovirus Type 7 Infection.

Epidemiologic reports suggest that the most severe or fatal adenoviral disease in children might be associated with human adenovirus (HAdV) type 7. However, the pathogenesis of HAdV-7-induced severe disease remains poorly understood. HAdV-3 and HAdV-7 replication kinetics and the host response to infection were compared using ex vivo human lung tissue cultures. Furthermore, cytokine and chemokine levels and the presence of adenovirus DNA in the serum of hospitalized children infected with HAdV-7 (n = 65) or HAdV-3 (n = 48) were measured (using a multiplex immunoassay and Taqman real-time polymerase chain reaction, respectively). Among 471 HAdV-positive specimens, HAdV-3 or HAdV-7 was the most prevalent genotype during 2014-2016 or 2018, respectively. The incidence of severe pneumonia was higher in HAdV-7-infected than in HAdV-3-infected individuals (30.1% vs 4.5%, respectively). HAdV-7 replicated more efficiently than HAdV-3 ex vivo. Interferon-induced protein 10, interleukin 6, and monocyte chemoattractant protein 1 levels were significantly higher in HAdV-7-infected than in HAdV-3-infected children. Adenovirus DNA was detected in serum samples from 40% and 4.2% of HAdV-7- and HAdV-3-infected children, respectively. Furthermore, viremia was strongly associated with severe clinical presentations. The pathogenesis of HAdV-7-induced severe disease was probably associated with high replication competence and hyperinflammatory responses. The detection of adenovirus DNA in blood may be useful in assessing risk for severe disease.

Impairment of endoxifen formation in tamoxifen-treated premenopausal breast cancer patients carrying reduced-function CYP2D6 alleles.

AimsTamoxifen is bioactivated to endoxifen by polymorphic CYP2D6‐dependent metabolism. Here, endoxifen levels were compared to CYP2D6 diplotypes, tentative target concentrations and side effects.MethodsIn total, 118 Swedish premenopausal breast cancer patients diagnosed 2006–2014, with on‐going postoperative tamoxifen treatment January 2017, were included. Biobanked DNA from peripheral blood was used for CYP2D6 genotyping by TaqMan real‐time polymerase chain reaction (CYP2D6*1, *3, *4, *5, *6, *9, *10, *41, *1xN). Plasma levels of tamoxifen and 3 major metabolites were quantified by liquid chromatography–tandem mass spectrometry. Clinical information on treatment and side effects was retrospectively obtained from medical records.ResultsIn the final analysis of 114 patients, a clear relationship between CYP2D6 genotype and plasma endoxifen levels was evident. Low endoxifen (1.6–5.2 ng/mL), i.e. below the suggested threshold for clinical efficacy, was found in all patients with 2 reduced‐function alleles, 2 null‐alleles, or a null/reduced‐function combination. CYP2D6*41 was the most common reduced‐function allele (82%) and 17 of 21 CYP2D6*41‐carriers exhibited a lower CYP2D6 activity than predicted from published guidelines. No difference in endoxifen levels was observed between carriers of 2 null‐alleles vs patients homozygous for CYP2D6*41 or the corresponding heterozygous combination (P = .338). In patients with endoxifen levels <5.9 ng/mL (36/114), side effects were either mild or absent. At higher endoxifen levels moderate‐to‐severe side effects were reported in a concentration‐dependent manner.ConclusionSignificantly reduced endoxifen levels were observed not only in all homozygous carriers of CYP2D6 null‐alleles, but also in carriers of 2 reduced‐function alleles. This finding may be highly relevant for future, genotype‐based dose considerations.

METTL3 polymorphisms and Wilms tumor susceptibility in Chinese children: A five-center case-control study.

Wilms tumor is a common pediatric tumor worldwide. Methyltransferase like 3 (METTL3) is a core gene of the N6 -methyladenosine (m6 A) modification that widely affects the transcription of tumor-related genes in eukaryotes. METTL3 has been extensively investigated in various tumors but not Wilms tumor. We describe a five-center case-control study with 414 patients and 1199 controls aiming to explore the associations between METTL3 polymorphisms (rs1061026 T>G, rs1061027 C>A, rs1139130 A>G and rs1263801 G>C) and Wilms tumor susceptibility. A TaqMan real-time polymerase chain reaction was performed for genotyping. Odds ratios (ORs) and 95% confidence intervals (CIs) were reported as evaluation indicators to determine any associations. Referring to the preliminary analysis results, protective genotypes were identified as rs1061026 TG/GG, rs1061027 CA/AA, rs1139130 GG and rs1263801 GC/CC. The children with three protective genotypes were less likely to develop Wilms tumor than children without protective genotypes (adjusted OR = 0.68, 95% CI = 0.46-0.999, p = 0.0496). Similarly, stratified analysis of the subgroup aged > 18 months, carrying 3 or 4 protective genotypes, was a protective factor for Wilms tumor compared to carrying 0-2 protective genotypes (adjusted OR = 0.59 95% CI = 0.39-0.91, p = 0.016). However, we did not observe any other significant results. The combined effect of METTL3 polymorphisms reduce Wilms tumor susceptibility in Chinese children. This conclusion requires further verification.

Identification of Novel Candidate Biomarkers for Oral Squamous Cell Carcinoma Based on Whole Gene Expression Profiling.

This study aimed to determine the whole gene expression profiles and to ascertain potential biomarkers for 22 oral squamous cell carcinoma (OSCC) among Thai patients using the Illumina Human HT-12, V4.0 Expression BeadChip array. Result indicated 2,724 differential expressed genes composed of 1,560 up-regulated and 1,164 down-regulated genes (unpaired t-test, p-value <0.05; fold change ≥2.0 and ≤2.0). The top 9 up-regulated genes were validated in 39 OSCC cases using TaqMan real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay. Among these, the up-regulation of peptidase inhibitor 3 (PI3) and keratin 17 (KRT17) genes was harbored in all 39 OSCC patients (100%). Likewise, statistical analysis indicated that gene expression in 8 selective genes including keratin 16 (KRT16), keratin 14 (KRT14), keratinocyte differentiation-associated protein (KRTDAP), keratin 6B (KRT6B), PI3, S100 calcium binding protein A7 (S100A7), stratifin (SFN) and keratin 5 (KRT5) was significantly associated with well differentiated OSCC (p-value <0.05). Moreover, high level of KRT17 protein was significantly associated with well differentiated OSCC compared to moderately OSCC (p-value = 0.041). Notably, using nested-PCR analysis indicated all OSCC cases in this study were HPV-free. Especially, KRTDAP, PI3, SFN mRNA expression were first reported among patients with OSCC. Conclusion, the whole transcript expression study and TaqMan real-time qRT-PCR assay were relevant regarding the increase in gene expression in OSCC. In addition, the up-regulation of PI3 and KRT17 might constitute potential candidate molecular biomarkers to diagnose patients with OSCC.

Detection of pork in meatballs using probe TaqMan Real-time Polymerase Chain Reaction

This study aimed to develop a TaqMan Real-Time Polymerase Chain Reaction method, using a novel primer for detection of pork adulteration in meatballs. The study is important as it described a TaqMan method for product adulteration analysis. TaqMan is known to have a more specific result compared to SYBR green analytical method. Assay in the study combined species-specific primers and TaqMan probes to targeting 153 bp fragment of D-loop mitochondrial region of pork. A specificity test was conducted on fresh tissues of pork, beef, chicken, wild boar, dog, and mouse. Meatballs as samples were prepared from a mixture of pork-beef and wild boar-beef with concentrations as follows: 5%, 10%, 25%, 75%, 90%, and 100%. The linearity and sensitivity of the method were performed by measuring the amplification curve from the dilution series, namely 1000, 200, 100, 10, 5, 1, and 0.5 pg/μL of DNA, extracted from 100% pork meatballs. A repeatability test was conducted as many as six repetitions on 100% pork and 100% wild boar meatballs. This study showed that mitochondrial D-loop species-specific primers and TaqMan probes could identify the DNA of pork and wild boar on the fresh tissues. Additionally, it also resulted in a threshold cycle (Ct) of 17.02 and 17.95 for pork, 22.22 for wild boar, while the negative result for others. The detection limit has shown 5 pg in the meatball formulation. The Relative Standard Deviation (RSD) of repeatability was 1.936% for pork, while 2.140% for wild boar. The developed method was also applied to analyzing commercial meatballs. A TaqMan real-time PCR analytical method using specific primer targeting on 153 bp fragment of the D-loop mitochondrial region could be applied as a standard method for identifying pork and wild boar in food samples intended for halal authentication studies

A MicroRNA Signature in Acute Coronary Syndrome Patients and Modulation by Colchicine

Circulating microRNAs (miRNAs) may play a pathogenic role in acute coronary syndromes (ACS). It is not yet known if miRNAs dysregulated in ACS are modulated by colchicine. We profiled miRNAs in plasma samples simultaneously collected from the aorta, coronary sinus, and right atrium in patients with ACS. A total of 396 of 754 miRNAs were detected by TaqMan real-time polymerase chain reaction from EDTA-plasma in a discovery cohort of 15 patients (n = 3 controls, n = 6 ACS standard therapy, n = 6 ACS standard therapy plus colchicine). Fifty-one significantly different miRNAs were then measured in a verification cohort of 92 patients (n = 13 controls, n = 40 ACS standard therapy, n = 39 ACS standard therapy plus colchicine). Samples were simultaneously obtained from the coronary sinus, aortic root, and right atrium. Circulating levels of 30 of 51 measured miRNAs were higher in ACS standard therapy patients compared to controls. In patients with ACS, levels of 12 miRNAs (miR-17, -106b-3p, -191, -106a, -146a, -130a, -223, -484, -889, -425-3p, -629, -142-5p) were lower with colchicine treatment. Levels of 7 of these 12 miRNA were higher in ACS standard therapy patients compared to controls and returned to levels seen in control individuals after colchicine treatment. Three miRNAs suppressed by colchicine (miR-146a, miR-17, miR-130a) were identified as regulators of inflammatory pathways. MicroRNAs were comparable across sampling sites with select differences in the transcoronary gradient of 4 miRNA. The levels of specific miRNAs elevated in ACS returned to levels similar to control individuals following colchicine. These miRNAs may mediate ACS (via inflammatory pathways) or increase post-ACS risk, and could be potentially used as biomarkers of treatment efficacy.

A Real-Time PCR Method for the Authentication of Common Cuttlefish (Sepia officinalis) in Food Products.

Cephalopods are very relevant food resources. The common cuttlefish (Sepia officinalis) is highly appreciated by consumers and there is a lack of rapid methods for its authentication in food products. We introduce a new minor groove binding (MGB) TaqMan real-time PCR (Polymerase Chain Reaction) method for the authentication of S. officinalis in food products to amplify a 122 base pairs (bp) fragment of the mitochondrial COI (Cytochrome Oxidase I) region. Reference and commercial samples of S. officinalis showed a threshold cycle (Ct) mean of 14.40, while the rest of the species examined did not amplify, or showed a significantly different Ct (p < 0.001). The calculated efficiency of the system was 101%, and the minimum DNA quantity detected was 10−4 ng. No cross-reactivity was detected with any other species, thus, the designed method differentiates S. officinalis from other species of the genus Sepia and other cephalopod species and works for fresh, frozen, grilled, cooked and canned samples of Sepia spp. The method has proved to be reliable and rapid, and it may prove to be a useful tool for the control of fraud in cuttlefish products.

Leptospira Detection in Cats in Spain by Serology and Molecular Techniques.

Leptospirosis is the most neglected widespread zoonosis worldwide. In Spain, leptospirosis reports in people and animals have increased lately. Cats can become infected with Leptospira, as well as be chronic carriers. The aim of this study was to determine serological antibody prevalence against Leptospira sp., blood DNA, and shedding of DNA from pathogenic Leptospira species in the urine of cats in Spain. Microagglutination tests (MAT) and blood and urine TaqMan real-time polymerase chain reaction (PCR) were performed. Leptospira antibodies were detected in 10/244 cats; with 4.1% positive results (95% confidence interval (CI): 2.1–7.18%). Titers ranged from 1:20 to 1:320 (serovars Ballum; Bataviae; Bratislava; Cynopteri; Grippotyphosa Mandemakers; Grippotyphosa Moskva; Pomona; and Proechimys). The most common serovar was Cynopteri. Blood samples from 1/89 cats amplified for Leptospira DNA (1.12%; 95% CI: 0.05–5.41%). Urine samples from 4/232 cats amplified for Leptospira DNA (1.72%; 95% CI: 0.55–4.10%). In conclusion free-roaming cats in Spain can shed pathogenic Leptospira DNA in their urine and may be a source of human infection. Serovars not previously described in cats in Spain were detected; suggesting the presence of at least 4 different species of pathogenic leptospires in the country (L. borgpetersenii; L. interrogans; L. kirschneri; and L. noguchii).

Human cytomegalovirus infection in Iranian glioma patients correlates with aging and tumor aggressiveness.

Human cytomegalovirus (HCMV), as a ubiquitous and opportunistic virus, is a matter forconsideration in broad-spectrum diseases, specifically in immunocompromised individuals. In recent decades, many studies that have evaluated the role of HCMV in inflammation and malignancies, especially in high-grade gliomas, have reported inconsistent results. Thus, this study was conducted to analyze 97 primary gliomas for human CMV UL83 gene and protein through TaqMan real-time polymerase chain reaction and immunohistochemistry, respectively. The results were positive for the UL83 gene and pp65 protein in 71% and 24% of samples, respectively. The frequency of HCMV was significantly higher in glioblastomas than other glioma grades (P < .01 and P < .05 for the UL83 gene and protein, respectively). In addition, the association between the prevalence of HCMV and aging strengthened the virus reactivation hypothesis in gliomas. In conclusion, a high frequency of HCMV infection was found in gliomas that correlated with tumor aggressiveness and age. This study recommends a thorough investigation to determine HCMV infection in gliomas to improve the existing knowledge of its rolein glial tumors, its prognostic value, and possible efficient antiviral target therapy.

AR420626, a selective agonist of GPR41/FFA3, suppresses growth of hepatocellular carcinoma cells by inducing apoptosis via HDAC inhibition.

Background:Hepatocellular carcinoma (HCC) is a major cause of cancer death worldwide and establishment of new chemotherapies for HCC is urgently needed. GPR41 [free fatty acid receptor 3 (FFA3)] is a G protein-coupled receptor for short chain fatty acids, including acetate, propionate, and butyrate. In our previous study, we showed that propionate enhances the cytotoxic effect of cisplatin in HCC cells and that this mechanism is dependent on inhibition of histone deacetylases (HDACs) via GPR41/FFA3. However, the antitumor action of GPR41/FFA3 has not been elucidated.Methods:In this study, we examined AR420626 as a GPR41-selective agonist in HepG2 and HLE cells. Nude mice were used for HepG2 xenograft studies. The apoptotic effect of AR420626 was evaluated using flow cytometry analysis. Expression of apoptosis-related proteins and HDACs was evaluated by Western immunoblot. Gene silencing of HDAC 3/5/7 and GPR41 was performed using small interfering RNA. Expression of TNF-α mRNA was evaluated by TaqMan real-time polymerase chain reaction.Results:We found that AR420626, a selective GPR41/FFA3 agonist, suppressed growth of HepG2 xenografts and inhibited proliferation of HCC cells by inducing apoptosis. AR420626 induced proteasome activation through mTOR phosphorylation, which reduced HDAC proteins, and then increased expression of TNF-α.Conclusion:AR420626, a selective GPR41/FFA3 agonist, may be a candidate as a therapeutic agent for HCC.