Search

Amplification Refractory Mutation System (ARMS) is the most popular technology for EGFR gene mutation analysis in China. Cutoff Ct or ΔCt values were used to differentiate low mutation abundance cases from no mutation cases. In this study, all of 359 NSCLC samples were tested by ARMS. Seventeen samples with larger Ct or ΔCt than cutoff values were retested by PCR-sequencing. TKI treatment responses were monitored on the cases with ARMS negative and PCR-sequencing positive results. One exon 18 G719X case, 67 exon 19 deletion cases, 2 exon 20 insertion cases, 1 exon 20 T790M case, 60 exon 21 L858R cases, 5 exon 21 L861Q cases and 201 wild type cases were identified by ARMS. Another 22 cases were evaluated as wild type but had later amplification fluorescent curves. Seventeen out of these 22 cases were retested by PCR-sequencing. It turns out that 3 out of 3 cases with exon 19 deletion later amplifications, 2 out of 2 cases with L858R later amplifications and 4 out of 12 cases with T790M later amplifications were identified as mutation positive. Two cases with exon 19 deletion and L858R respectively were treated by TKI and got responses. Our study indicated that PCR-sequencing might be a complementary way to confirm ARMS results with later amplifications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The continued spread of Candida auris in healthcare facilities has increased the demand for widely available screening to aid in containment and inform treatment options. Current methods of detection can be unreliable and require bulky and expensive instruments to lyse and identify fungal pathogens. Here, we present a quick, low-cost, instrument-free method for lysis of C. auris suitable for streamlined sample processing with polymerase chain reaction (PCR) detection. Chemical, thermal, and bead beating lysis techniques were evaluated for lysis performance and compatibility with nucleic acid extraction and downstream PCR reactions. Using only 10 s of manual shaking with glass beads, this method demonstrated a limit of detection (LOD) of C. auris at 500 colony forming units per mL, a 20-fold improvement compared to the LOD without manual shaking, and a 60-fold reduction in time compared to common fungal lysis kits, all while maintaining repeatability and reproducibility across multiple users. This work highlights a simple method for increasing sensitivity and reducing turnaround time of PCR-based C. auris detection and exhibits promise for integration into point-of-care platforms towards real-time triage of colonized patients. [ABSTRACT FROM AUTHOR]

Paired-like homeobox 2B (PHOX2B) is a highly sensitive and specific biomarker for diagnosing neuroblastoma, as well as detecting minimal residual disease in neuroblastoma. The clinical significance of PHOX2B expression in bone marrow (BM) and peripheral blood (PB) samples of newly diagnosed patients with very low-, low- and intermediate-risk neuroblastoma remains unknown, to the best of our knowledge. The expression level of PHOX2B in paired BM and PB samples of patients with newly diagnosed neuroblastoma was validated using reverse transcription-quantitative polymerase chain reaction (RTqPCR). Among the 132 patients, 26 exhibited a positive PHOX2B expression BM (19.7%) and 11 in PB (8.3%) samples. PHOX2B was highly expressed in BM and PB samples from patients aged <18 months, with International Neuroblastoma Risk Group Staging System stages M and MS, 1p loss of heterozygosity, and high levels of lactate dehydrogenase, serum ferritin and neuron-specific enolase (p < 0.05). In all eligible patients, the 2-year event-free survival (EFS) and overall survival (OS) rates were 94.7 ± 2.0% and 97.7 ± 1.3%, respectively. However, the 2-year EFS rates were significantly decreased to 76.9 ± 8.3% and 63.6 ± 14.5% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). Similarly, the 2-year OS rates were also decreased to 88.5 ± 6.3% and 81.8 ± 11.6% in patients with a positive PHOX2B expression in BM and PB samples, respectively (p < 0.05). In conclusion, a positive PHOX2B expression in BM and PB samples at diagnosis had a strong adverse prognostic effect on patients with non-high-risk neuroblastoma. [ABSTRACT FROM AUTHOR]

In our research, we explored the relationship between Keratin 26 and the regulation of fine hair, BMP signaling pathway, MT, FGF5, and IGF-I. The result of hybridization in situ revealed that Keratin 26 was specially expressed in cortex of skin hair follicles; the result of immunohistochemistry indicated that Keratin 26 was expressed in internal root sheath, external root sheath. Then, Real-time quantitative PCR results showed that relative expressive quantity of Keratin 26 was 1.08 or 3.3 × greater in secondary follicle than primary follicle during anagen or catagen; the difference during anagen was not remarkable (p>0.05), however, that of catagen was extremely significant (p<0.01). Relative expressive quantity of Keratin 26 increased during telogen; the difference was extremely significant (p<0.01). Moreover, after Noggin expression interference using RNAi technology, we found that relative expressive quantity of Keratin 26 extremely remarkably declined (p<0.01); after K26 overexpression, we found that relative expressive quantity of Noggin extremely remarkably increased (p<0.01). We detected expressive quantity change of Keratin 26 and Keratin 26 using Real-time quantitative PCR and immunofluorescence technologies after fibroblasts were treated with MT, FGF5 or IGF-I; the results indicated that MT and FGF5 played a positive role in Keratin 26 and Keratin 26 expression, IGF-I played a negative role in Keratin 26 expression, positive role in Keratin 26 expression. The results above showed that Keratin 26 could inhibit cashmere growth, and was related to entering to catagen and telogen of hair follicles; Keratin 26 and BMP signaling pathway were two antagonistic pathways each other which could inhibit growth and development of cashmere; MT, FGF5 and IGF-I could affect expression of Keratin 26 and Keratin 26, and Keratin 26 was one of the important pathways that MT induced cashmere production in advance, FGF5 regulated cashmere growth and IGF-I promoted cashmere growth and development. [ABSTRACT FROM AUTHOR]

DNA barcoding is a fast-developing technique to identify species by using short and standard DNA sequences. Universal selection of DNA barcodes in ferns remains unresolved. In this study, five plastid regions (rbcL, matK, trnH-psbA, trnL-F and rps4-trnS) and eight nuclear regions (ITS, pgiC, gapC, LEAFY, ITS2, IBR3_2, DET1, and SQD1_1) were screened and evaluated in the fern genus Adiantum from China and neighboring areas. Due to low primer universality (matK) and/or the existence of multiple copies (ITS), the commonly used barcodes matK and ITS were not appropriate for Adiantum. The PCR amplification rate was extremely low in all nuclear genes except for IBR3_2. rbcL had the highest PCR amplification rate (94.33%) and sequencing success rate (90.78%), while trnH-psbA had the highest species identification rate (75%). With the consideration of discriminatory power, cost-efficiency and effort, the two-barcode combination of rbcL+ trnH-psbA seems to be the best choice for barcoding Adiantum, and perhaps basal polypod ferns in general. The nuclear IBR3_2 showed 100% PCR amplification success rate in Adiantum, however, it seemed that only diploid species could acquire clean sequences without cloning. With cloning, IBR3_2 can successfully distinguish cryptic species and hybrid species from their related species. Because hybridization and allopolyploidy are common in ferns, we argue for including a selected group of nuclear loci as barcodes, especially via the next-generation sequencing, as it is much more efficient to obtain single-copy nuclear loci without the cloning procedure. [ABSTRACT FROM AUTHOR]

The article focuses on a study related to the selective deletion of the major alpha globin regulatory element (MCS-R2) causing alpha-thalassaemia. It mentions nondeletional and deletional mutations of Hemoglobin alpha (HBA1) and Hemoglobin, alpha 2 (HBA2) excluded by direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) in the carriers. It also mentions chromosome mapping in patients leads to the identification of deletions located far upstream.

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61-vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection. [ABSTRACT FROM AUTHOR]

Background: Although previous studies have demonstrated an association between ABO blood group and the risk of gastric cancer (GC), only one study has identified these associations using the ABO genotype; however, that study did not evaluate sex differences in this association. The aim of the present study was to investigate whether there are sex-specific differences in the ABO genotype-associated risk of GC. In addition, we explored the association of the ABO genotype and the clinicopathologic characteristics of GC in a Korean population. Methods: We conducted a large-scale case-control study of 3245 GC patients (2204 males, 1041 females) and 1700 controls (821 males, 879 females). The ABO genotype was determined by multicolor real-time polymerase chain reaction (PCR) using displacing probes. Results: As compared with genotype OO, genotypes AA and AO in females, but not in males, were associated with a significantly increased risk of GC (odds ratio [OR] 1.56 and 95 % confidence interval [CI] 1.08-2.26 for AA; OR 1.57 and 95 % CI 1.21-2.03 for AO). In a subgroup analysis, blood group A had a significantly increased risk of diffuse-type GC (OR 2.00, 95 % CI 1.43-2.78), but not of intestinal-type (OR 1.31, 95 % CI 0.96-1.79) or mixed-type GC (OR 1.43, 95 % CI 0.92-2.24). Conclusion: The ABO genotypes AA and AO were significantly associated with GC only in females and only for diffuse-type GC. These data suggest that the association between ABO blood group and GC risk may differ according to sex and histological type. [ABSTRACT FROM AUTHOR]

Human serum paraoxonase 1 (PON1), a calcium-dependent ester hydrolase, protects against the oxidative modification of low-density lipoprotein (LDL) and is a major anti-atherosclerotic component of high-density lipoprotein (HDL). Graptopetalum paraguayense, a folk herbal medicine commonly used in Taiwan, has antioxidative, anti-inflammatory, anti-hypertensive, and anti-atherogenic properties. The effects of G. paraguayense on the activity and/or expression of PON1 were examined using various extracts of the plant; extracts were made in water (GPWE), 50% ethanol (GP50E), and 95% ethanol (GP95E). Of these extracts, GP50E was found to be the most effective at increasing the function and expression of PON1 in a human hepatoma HepG2 cell line. Data from electrophoretic mobility shift assays and promoter-reporter luciferase analyses demonstrated that the DNA binding activity and transactivation ability of NF-κB were enhanced by GP50E. Treatment with NF-κB inhibitors, pyrrolidine dithiocarbamate, and BAY 11-7082 significantly attenuated GP50E-induced PON1 production and NF-κB transactivation activity. In addition, GP50E increased the levels of phosphorylated protein kinase B (PKB/AKT). Pharmacological inhibition of AKT by LY294002 effectively suppressed NF-κB activation and PON1 gene expression, suggesting that AKT was an upstream regulator of GP50E-mediated biological events. Overall, the results show that GP50E up-regulated PON1 gene expression via an AKT/NF-κB-dependent signaling pathway in human hepatoma HepG2 cells. This observation led to the conclusion that the anti-atherogenic characteristics of G. paraguayense are modulated, at least in part, via the up-regulation of hepatocyte PON1 gene expression. [ABSTRACT FROM AUTHOR]

Background: The common p53 codon 72 polymorphism has been investigated as a risk factor for cancer in different populations; however, the results have been inconsistent. This study investigated the risk of developing gastric or colorectal cancer associated with the p53 codon 72 polymorphism in a Korean population. Methods: We conducted a large-scale case-control study that included 2,213 gastric cancer patients; 1,829 colorectal cancer patients; and 1,700 healthy controls. Genotyping was performed with real-time polymerase chain reaction (PCR), using a TaqMan single-nucleotide polymorphism (SNP) genotyping assay. Results: The frequencies of Arg/Arg, Arg/Pro, and Pro/Pro genotypes of the p53 codon 72 polymorphism were 43.3, 42.0, and 13.0% in the gastric cancer patients; 40.5, 45.0, and 14.0% in the colorectal cancer patients; and 43.2, 45.6, and 11.2% in the controls, respectively. The Pro/Pro genotype was associated with an increased risk of gastric [age- and sex-adjusted odds ratio (OR) = 1.25, 95% confidence interval (CI) = 1.01-1.56, P = 0.04] and colorectal cancer (OR = 1.36, 95% CI = 1.07-1.72, P = 0.01). There were no significant interactions between the p53 codon 72 polymorphism and smoking or drinking. Conclusions: Our results suggest that the Pro/Pro genotype is associated with modest increases in the risks of gastric cancer and colorectal cancer in a Korean population. [ABSTRACT FROM AUTHOR]

The inhibin β ( INHBB) gene was studied as a candidate gene for the prolificacy of Small Tail Han and Hu sheep. According to the sequence of exon 1 and 2 of bovine INHBB gene, six pairs of primers were designed to detect single nucleotide polymorphisms of exon 1 and 2 of INHBB gene in both high (Small Tail Han and Hu sheep) and low prolificacy breeds (Dorset, Texel and German Mutton Merino sheep) by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). Three pairs of primers (primers 1-1, 1-2 and 1-3) were used to amplify the exon 1, and others (primers 2-1, 2-2 and 2-3) to the exon 2. Only the products amplified by primer 2-3 displayed polymorphism. For primer 2-3, three genotypes (AA, AB and BB) were detected in Hu sheep and only AA genotype in other breeds. In Hu sheep, frequency of AA, AB and BB genotypes was 0.636, 0.046 and 0.318, respectively. Sequencing revealed 276A > G mutation (based on the amplification region of primer 2-3) which did not cause any amino acid change because it lay in the 3′ untranslated region. The ewes with genotype BB had 0.58 ( P < 0.01) lambs more than those with AA in Hu sheep. [ABSTRACT FROM AUTHOR]

The article describes the development of a single-tube, one-step, real-time accelerated reverse transcription (RT)-loop-mediated isothermal amplification (LAMP) for the detection of Polvine teschovirus (PTV). It also notes that LAMP is a sensitive method for dinucleic acid (DNA) amplification. The results demonstrated that the test is simple, fast, accurate, and sensitive for the detection of PTV when 43 clinical samples were tested by the RT-LAM P method.

We report 3 cases of Queensland tick typhus (QTT) and 1 case of African tick bite fever in which the causative rickettsiae were detected by PCR of eschar and skin lesions in all cases. An oral mucosal lesion in 1 QTT case was also positive. [ABSTRACT FROM AUTHOR]

• HHV-6B triggers immune modulatory factors in MTLE. • Increased IL-1a, IL-2 and IL-7 mRNA levels are associated with early antigen expression. • IL-1a and IL-7 may be taken as indirect biomarker of HHV-6B infection in CSF. • Co-localization of HHV-6B antigen with astrocytes and oligodendrocytes. Background: Human herpes virus-6B (HHV-6B) was suggested as an important etiologic factor of mesial temporal lobe epilepsy, while the mechanism is still unknown. Here, we aimed to analyze antigens representing latent, early and late HHV-6B infection and the association with inflammatory cytokines in brain tissue and cerebral spinal fluid (CSF) from MTLE patients with HHV-6B-positivity. Methods: Nested polymerase chain reaction (nPCR), real-time PCR, immunohistochemistry (IHC) and suspension bead array for cytokines were performed. Results : Nested polymerase chain reaction (nPCR) in brain tissue revealed HHV-6B DNA in 19 of 49 MTLE patients (39%) and 1 of 19 controls (5%) (P < 0.001), but not in CSF. ICH showed HHV-6B early antigen (P41) positivity in 3 patients (6%), late antigen (gp116/54/64) positivity in 5 patients (10%), latent antigen (U94) positivity in 8 patients (16%), and multiple antigen (early and late or/and latent) positivity in 9 patients (18%). None of these HHV-6B related proteins were found positive in control brain tissue. PCR revealed significant up-regulation of IL-1a, IL-2 and IL-7 mRNA levels in the brain tissue from MTLE patients expressing early antigens compared to those expressing late, latent, multiple antigens, negative antigens and the controls. Suspension bead array of the CSF confirmed significant up-regulation of IL-1a and IL-7 protein expression from MTLE patients expressing early antigens compared to the other groups. Conclusions: Our finding suggests HHV-6B is a common etiologic agent of MTLE. Different virus life cycle may play an important modifying role in inflammatory biology that warrants further investigation. Though virus DNA is difficult detected in CSF, up-regulation of IL-1a and IL-7 in CSF indicates the two cytokines may be taken as indirect biomarker of HHV-6B infection. [ABSTRACT FROM AUTHOR]

• An all covariates-based association model was built to explain the risk of CAD. • The model incorporated both a gene-score and 8 lifestyle/clinical risk factors. • The gene-score was derived from ALDH2 , ADCY3 and BCMO1 polymorphisms. • The findings may provide implications for CAD pathogenesis in Chinese Han population. To investigate associations of genetic and environmental factors with coronary artery disease (CAD), we collected medical reports, lifestyle details, and blood samples of 2113 individuals, and then used the polymerase chain reaction (PCR)-ligase detection reaction (LDR) to genotype the targeted 102 SNPs. We adopted elastic net algorithm to build an association model that considered simultaneously genetic and lifestyle/clinical factors associated with CAD in Chinese Han population. In this study, we developed an all covariates-based model to explain the risk of CAD, which incorporated 8 lifestyle/clinical factors and a gene-score variable calculated from 3 significant SNPs (rs671, rs6751537 and rs11641677), attaining an area under the curve (AUC) value of 0.71. It was found that, in terms of genetic variants, the AA genotype of rs671 in the additive (adjusted odds ratio (OR) = 2.51, p = 0.008) and recessive (adjusted OR = 2.12, p = 0.021) models, the GG genotype of rs6751537 in the additive (adjusted OR = 3.36, p = 0.001) and recessive (adjusted OR = 3.47, p = 0.001) models were associated with increased risk of CAD, while GG genotype of rs11641677 in additive model (adjusted OR = 0.39, p = 0.044) was associated with decreased risk of CAD. In terms of lifestyle/clinical factors, the history of hypertension (unadjusted OR = 2.37, p < 0.001) and dyslipidemia (unadjusted OR = 1.82, p = 0.007), age (unadjusted OR = 1.07, p < 0.001) and waist circumference (unadjusted OR = 1.02, p = 0.05) would significantly increase the risk of CAD, while height (unadjusted OR = 0.97, p = 0.006) and regular intake of chicken (unadjusted OR = 0.78, p = 0.008) reduced the risk of CAD. A significant interaction was found between rs671 and dyslipidemia (the relative excess risk due to interaction (RERI) = 3.36, p = 0.05). In this study, we constructed an association model and identified a set of SNPs and lifestyle/clinical risk factors of CAD in Chinese Han population. By considering both genetic and non-genetic risk factors, the built model may provide implications for CAD pathogenesis and clues for screening tool development in Chinese Han population. [ABSTRACT FROM AUTHOR]

Abstract: Porcine teschovirus (PTV), the pathogen of porcine polioencephalomyelitis, is a member of the family Picornaviridae. In this study, a new PTV strain (designated as JF613) was isolated from pigs in China. It was confirmed by the specific CPE on susceptible cells, RT-PCR and nucleotide sequencing. Analysis of its amino acids sequence of complete polyprotein indicated that the isolate belongs to serotype 2. Genetic recombination is a well-known phenomenon for picornavirus which has been demonstrated in many other members of the family, but it remains so far unclear whether recombination occurs in PTV. To detect possible recombination events, 30 sequences of complete coding regions of PTV strains accessible in GenBank were examined. Putative recombinant sequence was identified with the use of SimPlot program. The result showed that the genomic sequence of our isolate exhibited highest similarities with strains of serotypes 2 and 5, respectively, in two crossover regions, suggesting the recombination event in PTV. Then the mosaic structure of viral genome was confirmed by bootscanning and genetic algorithm for recombination detection (GARD). This represents the first PTV-2 isolate in China. Furthermore, our study provided the first evidence of natural recombination in PTV and indicated that homologous recombination may be a driving force in PTV evolution. [Copyright &y& Elsevier]

Abstract: In this study, the sheep CIB4 cDNA was cloned from the small tail Han sheep by RT-PCR and RACE (rapid amplification of cDNA ends), and CIB4 cDNA and amino acid sequence were analyzed. Our results showed that the sheep CIB4 gene expressed two alternatively spliced variants L-CIB4 (long CIB4) and S-CIB4 (short CIB4). Sequence analysis indicated that the sheep CIB4 cDNA cloned (L-CIB4) was 745-bp in length (GenBank accession number: FJ039532) with 185 amino acids residues. The sheep CIB4 cDNA showed more than 72% of sequence identity, at the nucleotide level, to its equivalents in cattle, horse, chimpanzee, humans, mice and rats, while at the deduced protein level, the value increased to 79.6%. Semi-quantitative RT-PCR using total RNA from different tissues showed that CIB4 has a strong tissue-specific expression pattern in sheep. L-CIB4 expression level was shown to be no different in small tail Han sheep and the Dorset ram, but both were significantly different from the Texel (P <0.05). Surprisingly, the short spliced form, S-CIB4, could only be detected in small tail Han sheep, suggesting that CIB4 may be linked in some way to the high fecundity of this breed. [Copyright &y& Elsevier]