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Introduction: The global public health concern of Staphylococcus aureus (S. aureus) infection and its spread, particularly methicillin-resistant Staphylococcus aureus (MRSA), is exacerbated by the emergence of mec-genes, which are considered markers of MRSA. The study aims to isolate MRSA in clinical isolates and detect mec-genes using molecular assays, thereby enhancing infection control programs. Materials and Methods: The study involved 381 clinical samples processed by standard laboratory procedures and PCR was performed targeting 16S rRNA gene as a reference and then with nuc-gene to detect Staphylococcus aureus. Further, MRSA was confirmed by PCR (Polymerase Chain Reaction) amplification of S. aureus isolates by subtyping mecA and mecC gene. Sensitivity and specificity of phenotypic methods was calculated using mecA gene PCR as the gold standard. Results: In the present study, 65.85% clinical isolates were found to have similar homology with Staphylococcus aureus. Uniplex PCR results showed that all 162 isolates with S. aureus-like phenotypes were detected as MRSA carrying mecA gene, but none of them was found to carry mecC gene for MRSA. P-value for both the methods was <0.001, which is considered to be highly significant in statistical analysis. Conclusion: The study found that PCR detection of nuc and mec genes is effectively identifies MRSA from clinical isolates, aiding in infection prevention and recommending its use for confirmatory testing. The study recommends using PCR as a confirmatory test method for MRSA detection to prevent the spread of resistant strains in hospitals and communities. [ABSTRACT FROM AUTHOR]

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p<0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p<0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping. [ABSTRACT FROM AUTHOR]

Introduction. Infections due to extended spectrum β-lactamase producing Enterobacteriaceae are on the rise. They pose serious public health problems due to their resistance to large number of antibiotics. However, little is known about the genotypes of ESBL from Nepal. Therefore, the study presents results of phenotypic and molecular characterization of ESBL producing Escherichia coli and Klebsiella spp. isolated from various clinical specimens in a tertiary care teaching hospital of Nepal. Methods. A total of 172 Enterobacteriaceae clinical isolates recovered from various clinical specimens were analyzed for their antibiotic susceptibility test. Detection of ESBLs was carried out using combination disk test and multiplex PCR for their genotypes (CTX-M, SHV, and TEM). Results. Out of 172 clinical isolates, 70 (40.6%) of them were found ESBL producers. The major source of ESBL producers was urinary tract samples and the highest ESBL production was observed in Escherichia coli (46.5%). Among ESBL genotypes, CTX-M (91.4%) was most predominant, followed by TEM (65.7%) and SHV (11.4%) in both of the isolates. Conclusions. High level of drug resistance and ESBL production was observed among the clinical isolates. There is a need for longitudinal and nationwide surveillance for drug resistance in clinical isolates and antimicrobial stewardship is necessary to guide the appropriate and judicious antibiotic use. [ABSTRACT FROM AUTHOR]

The 41 kD flagellin of Borrelia burgdorferi (B. burgdorferi) is a major component of periplasmic flagellar filament core and a good candidate for serodiagnosis in early stage of Lyme disease. Here, we chose 89 B. burgdorferi strains in China, amplified the gene encoding the 41 kD flagellin, and compared the sequences. The results showed that genetic diversity presented in the 41 kD flagellin genes of all 89 strains among the four genotypes of B. burgdorferi, especially in the genotype of B. garinii. Some specific mutation sites for each genotype of the 41 kD flagellin genes were found, which could be used for genotyping B. burgdorferi strains in China. Human B-cell epitope analysis showed that thirteen of 15 nonsynonymous mutations occurred in the epitope region of 41 kD flagellin and thirty of 42 B-cell epitopes were altered due to all 13 nonsynonymous mutations in the epitope region, which may affect the function of the antigen. Nonsynonymous mutations and changed human B-cell epitopes exist in 41 kD flagellin of B. burgdorferi sensu lato strains; these changes should be considered in serodiagnosis of Lyme disease. [ABSTRACT FROM AUTHOR]

Schistosomiasis is a neglected tropical disease of humans, and it is considered to be the second most devastating parasitic disease after malaria. Eggs produced by normally developed female worms are important in the transmission of the parasite, and they responsible for the pathogenesis of schistosomiasis. The tumor suppressor gene lethal giant larvae (lgl) has an essential function in establishing apical-basal cell polarity, cell proliferation, differentiation, and tissue organization. In our earlier study, downregulation of the lgl gene induced a significant reduction in the egg hatching rate of Schistosoma japonicum (Sj) eggs. In this study, the Sjlgl gene was used as a vaccine candidate against schistosomiasis, and vaccination achieved and maintained a stable reduction of the egg hatching rate, which is consistent with previous studies, in addition to reducing the worm burden and liver egg burden in some trials. [ABSTRACT FROM AUTHOR]

A novel thermostable glucoamylase cDNA without starch binding domain (SBD) of Aspergillus flavus NSH9 was successfully identified, isolated, and overexpressed in Pichia pastoris GS115. The complete open reading frame of glucoamylase from Aspergillus flavus NSH9 was identified by employing PCR that encodes 493 amino acids lacking in the SBD. The first 17 amino acids were presumed to be a signal peptide. The cDNA was cloned into Pichia pastoris and the highest expression of recombinant glucoamylase (rGA) was observed after 8 days of incubation period with 1% methanol. The molecular weight of the purified rGA was about 78 kDa and exhibited optimum catalytic activity at pH 5.0 and temperature of 70°C. The enzyme was stable at higher temperature with 50% of residual activity observed after 20 min at 90°C and 100°C. Low concentration of metal (Mg++, Fe++, Zn++, Cu++, and Pb++) had positive effect on rGA activity. This rGA has the potential for use and application in the saccharification steps, due to its thermostability, in the starch processing industries. [ABSTRACT FROM AUTHOR]

An increasing body of evidence has indicated that polymorphisms in the miRNA binding site of target gene can alter the ability of miRNAs to bind their target genes and modulate the risk of cancer. We aimed to investigate the association between a miR-520a binding site polymorphism rs141178472 in the PIK3CA 3′-UTR and the risk of colorectal cancer (CRC) in a Chinese Han population. The polymorphism rs141178472 was analyzed in a case-control study, including 386 CRC patients and 394 age- and sex-matched controls; the relationship between the polymorphism and the risk of colorectal cancer was examined. Individuals carrying the rs141178472 CC genotype or C allele had an increased risk of developing CRC (CC versus TT, OR (95% CI): 1.716 (1.084–2.716), P=0.022; C versus T, OR (95% CI): 1.258 (1.021–1.551), P=0.033). Furthermore, the expression of PIK3CA was detected in the peripheral blood mononucleated cell of CRC patients, suggesting that mRNA levels of PIK3CA might be associated with SNP rs141178472. These findings provide evidence that a miR-520a binding site polymorphism rs141178472 in the PIK3CA 3′-UTR may play a role in the etiology of CRC. [ABSTRACT FROM AUTHOR]

Objective: To determine whether there is a relationship between genetic polymorphisms of glutathione S-transferase zeta 1 (GSTZ1) and gastric cancer. The contribution of GSTZ1 genotypes to susceptibility to the risk of gastric cancer (GC) is still unclear. Methods: Using a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method in an ethnic Turkish population, we examined the frequency of the GSTZ1 gene polymorphism in patients with GC patients (n = 73) and control individuals (n = 80). Results: For GSTZ1 A94G polymorphism, in the group of patients with the GC, the frequency of the GG genotype was quite a bit higher in comparison with that of the control group; however, this increase was not statistically significant. For the GSTZ1 A124G polymorphism, the GSTZ1 heterozygous genotype (AG) occurred more frequently in GC patients than in controls; however, it was not associated with risk of developing GC. We found no significant association between the A94G or A124G variants of the GSTZ1 gene and risk of gastric cancer. Conclusions: Our data indicate no association between GSTZ1 genotypes and risk of gastric cancer. Despite its marked decline in many industrialized countries, gastric cancer remains the most common cause of death by cancer in areas such as Japan, Turkey, and South America. Gastric cancer (GC) is a disease of complex etiology that involves intimately interconnected infectious, dietary, environmental, and genetic factors. Although it has been estimated that 67% of GCs could be prevented by implementing lifestyle changes, the fact that some individuals develop GC but others do not, despite exposure to similar potentially carcinogenic factors, suggests that genetic predisposition may also play an important role in the etiology of GC. [ABSTRACT FROM AUTHOR]

Background: Methylenetetrahydrofolate reductase (MTHFR) polymorphisms have recently raised the interest as a possible thrombophilic factors. Aims: We aimed to assess the frequency of the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms in idiopathic venous thromboembolism (VTE) in a Romanian population and the associated risk of VTE. Study Design: We performed a case-control transversal study including 90 patients diagnosed with VTE and 75 sex- and age-matched controls. Methods: MTHFR C677T and A1298C polymorphisms were detected using PCR-RFLP method. Results: The homozygous MTHFR 677TT genotype, present in 18.8% of patients with VTE versus 6.6% of controls, was significantly associated with VTE (p= 0.021, OR= 3.26, 95%CI (1.141-9.313)). The heterozygous MTHFR A1298C genotype, presenting the highest prevalence in the VTE group (34.4%) as well as in controls (37.3%), was not associated with VTE (p=0.7). No associations were found for heterozygous MTHFR C677T (with a frequency of 32.2% in VTE and 37.3% in controls, p=0.492), respective homozygous MTHFR A1298C genotype (with a frequency of 1.1% in VTE and 2.6% in controls, p=0.456). Conclusion: Among MTHFR polymorphisms, only homozygosity for MTHFR 677TT may be considered a risk factor for VTE; the MTHFR A1298C polymorphism is not significantly associated with an increased risk of VTE. [ABSTRACT FROM AUTHOR]

Objective: To characterize the molecular features of seven extended-spectrum betalactamase-producing (ESBL) strains of Klebsiella pneumoniae in patients with bacteremia during a nosocomial outbreak in a neonatal unit in Lima, Peru. Methods: Genotyping was performed using Eric-PCR, Rep-PCR and pulsed field gel electrophoresis (PFGE) to evaluate clonality. Disk diffusion test showed that all seven strains were ESBL producing strains. Results: The three-genotyping methods showed that five out of seven strains were genetically related to a single clone. Conclusions: The study represents the first attempt to apply genotyping methods to study nosocomial outbreaks in neonatal units in Lima, and reveals clonal transmission of ESBL producing strains of K. pneumoniae. [ABSTRACT FROM AUTHOR]

We investigated postprandial changes in transcript abundance following a single satiating meal in juvenile Atlantic salmon (Salmo salar L.) (about 70 g body mass) following fasting for 1 week at 12°C. The expression of twenty-three growth-related genes was determined in fast myotomal muscle using quantitative real-time PCR at the following postprandial time points: − 12, 0, 1, 3, 6, 12, 24, 48 and 96 h. The gut was fullest 1–6 h after feeding and emptied within 48–96 h. IGF-I, MyoD1c, MRF4 and myf5 transcripts were sharply up-regulated within 1 h of refeeding and are promising candidate genes involved in a fast-response signalling system that regulates fish myotomal muscle growth. These genes clustered together with MyoD1b and suggest a coordinated regulation to favour resumption of myogenesis as an early response to feeding. Insulin-like growth factor (IGF)-II and the ubiquitin ligase MAFbx/atrogin-1 were initially down-regulated but restored to initial values after 12 h. It is also suggested that local production of IGF-I within the muscle might suppress catabolic pathways depressing MAFbx/atrogin-1. [ABSTRACT FROM PUBLISHER]

The purpose of this study was to isolate the coding sequence of the p24 protein (encoded by the gag gene) from sera collected from Iranian patients, to clone and express this sequence in Escherichia coli (E coli), and to assess the immunoreactivity of the expressed recombinant protein. The complete coding sequence for the Gag protein was isolated by nested real-time polymerase chain reaction {RT-PCR) from the serum of an infected individual and then cloned into a PTZ57R (T) vector. The resulting vector, PTZ-gag53-IR, was sequenced. Subsequently, the coding sequence of p24 protein was isolated by PCR from the PTZ-gag53-IR vector and inserted into a pET102/D-TOPO vector using TOPO directional cloning and then expressed in E coli in fusion with His-Patch (HP)-thioredoxin. The immunoreactivity of the expressed protein was tested by immunoblotting and enzyme-linked immunosorbent assay (ELISA). RT-PCR revealed a sequence belonging to the CRF35_AD subtype of the HIV-1 virus, which is highly prevalent in Iran and Afghanistan. Recombinant p24 protein from this isolate showed 100% sensitivity and 100% specificity in an antibody assay. [ABSTRACT FROM AUTHOR]

Introduction: In spite nearly 40% of the variability in blood pressure can be explained by genetic factors, the identification of genes associated to essential high blood pressure is difficult in populations where individuals have different genetic precedents; in these circumstances it is necessary to determinate whether the population is sub-structured because this can bias studies associated with this disease. Objectives: To determine the genetic structure of the population in Bucaramanga from genetic polymorphisms associated with the regulation of blood pressure: 448G>T, 679C>T y 1711C>T from the gene kinase 4 of the dopaminergic receptor linked to the protein G and Glu298Asp, -786T>C and the VNTR of the intron 4 of the gene of endothelial nitric oxide. Methodology: A sample of 552 unrelated individuals was studied through analysis of Restriction fragment length polymorphism. The allelic, haplotypic and genotypic frequencies were calculated, the Hardy-Weinberg equilibrium was determined and a molecular analysis of variance was performed to determine the genetic structure. Results: 38 Haplotypes were identified, with GCCTG4b as the most frequent (21.2%). The most diverse polymorphism was 448G>T with a frequency of 49.9% for heterozygous. The six polymorphisms were found in genetic equilibrium and genetic structure of populations was not evidenced (FST = 0,0038). Conclusion: The population studied does not present a genetic sub-structure and the polymorphisms analyzed were found in genetic equilibrium, this indicates that the population mixes randomly and there are no sub-groups capable of affecting the results of the association studies. [ABSTRACT FROM AUTHOR]

Background: It is difficult to study the mechanisms of the metabolic syndrome in humans due to the heterogeneous genetic background and lifestyle. The present study investigated changes in the gene and protein profiles in an animal model of the metabolic syndrome to identify the molecular targets associated with the pathogenesis and progression of obesity related to the metabolic syndrome. Methods: We extracted mRNAs and proteins from the liver tissues of 6- and 25-week-old spontaneously hypertensive/NIH -corpulent rat SHR/NDmcr-cp (CP), SHR/Lean (Lean) and Wistar Kyoto rats (WKY) and performed microarray analysis and two-dimensional difference in gel electrophoresis (2D-DIGE) linked to a matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS). Results: The microarray analysis identified 25 significantly up-regulated genes (P < 0.01; log10 > 1) and 31 significantly down-regulated genes (P < 0.01; log10 < ?1) in 6- and 25-week-old CP compared with WKY and Lean. Several of these genes are known to be involved in important biological processes such as electron transporter activity, electron transport, lipid metabolism, ion transport, transferase, and ion channel activity. MALDI-TOF/TOF MS identified 31 proteins with ±1.2 fold change (P < 0.05) in 6- and 25-week-old CP, compared with age-matched WKY and Lean. The up-regulated proteins are involved in metabolic processes, biological regulation, catalytic activity, and binding, while the down-regulated proteins are involved in endoplasmic reticulum stress-related unfolded protein response. Conclusion: Genes with significant changes in their expression in transcriptomic analysis matched very few of the proteins identified in proteomics analysis. However, annotated functional classifications might provide an important reference resource to understand the pathogenesis of obesity associated with the metabolic syndrome. [ABSTRACT FROM AUTHOR]

Background: Alpha-1 antitrypsin (AAT) deficiency is an inherited disease characterized by reduced levels of AAT in the serum. The 2 common genotypes of AAT deficiency are type Z (PiZ) and type S (PiS), which are associated with several malignancies. Methods: We assessed the AAT phenotype as well as genotypes Z and S in squamous cell carcinoma of the esophagus (SCCE) and their association with malignancy in Azeri patients of Iran. Alpha-1 antitrypsin phenotype identification was done using isoelectric focusing (IEF); its genotype was identified by RFLP. Results: The results indicated that the mean range of trypsin inhibitory capacity (TIC) and AAT nephlometry are significantly different in patients (P<0.05). In addition, while AAT levels are higher in the serum of patients they are not functional. This accounts for further dysfunction and reduction of its proper protease inhibitory activity in patients when compared with the healthy controls. Moreover, 97.3% of SCCE patients were homozygote for MM (PiMM), and only 2.7% were MS heterozygous. Neither of the PiZ and PiS genotypes were identified in the patients (P<0.05). Conclusion: Alpha-1 antitrypsin is a suitable prognostic rather than early stages diagnostic tumor marker in SCCE as its correlation with the disease could significantly be seen in the advanced stages of the esophageal tumorigenesis. As a tumor marker, AAT is highly sensitive but less tissue specific, thus its application is useful in combination with a panel of tumor markers in SCCE diagnosis, treatment, and follow up. [ABSTRACT FROM AUTHOR]