Aberrant methylation of N-methyl-D-aspartate receptor type 2B (NMDAR2B) in non-small cell carcinoma

<p>Abstract</p> <p>Background</p> <p>N-methyl-D-aspartate receptors (NMDAR) act as tumor suppressors of digestive malignancies. The expression and genetic methylation patterns of <it>NMDAR2B </it>in non-small cell lung cancer (NSCLC) are unknown.</p> <p>Methods</p> <p>The relationship between gene methylation and expression of <it>NMDAR2B </it>was analyzed in NSCLC cell lines (N = 9) and clinical tissues (N = 216). The cell lines were studied using RT-PCR and 5-aza-2'-deoxycytidine treatment, while the clinical tissues were examined by methylation specific real-time quantitative PCR and immunohistochemistry. Retrospective investigation of patient records was used to determine the clinical significance of <it>NMDAR2B </it>methylation.</p> <p>Results</p> <p><it>NMDAR2B </it>was silenced in five of the nine cell lines; 5-aza-2'-deoxycytidine treatment restored expression, and was inversely correlated with methylation. Aberrant methylation of <it>NMDAR2B</it>, detected in 61% (131/216) of clinical NSCLC tissues, was inversely correlated with the status of protein expression in 20 randomly examined tumors. Aberrant methylation was not associated with clinical factors such as gender, age, histological type, or TNM stage. However, aberrant methylation was an independent prognostic factor in squamous cell carcinoma cases.</p> <p>Conclusions</p> <p>Aberrant methylation of the <it>NMDAR2B </it>gene is a common event in NSCLC. The prognosis was significantly better for cases of squamous cell carcinoma in which <it>NMDAR2B </it>was methylated. It may have different roles in different histological types.</p

A standardised method for interpreting the association between mutations and phenotypic drug resistance inMycobacterium tuberculosis

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence. Raw genotype–phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance. We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6–90.9%), while for isoniazid it was 78.2% (77.4–79.0%) and their specificities were 96.3% (95.7–96.8%) and 94.4% (93.1–95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1–70.6%) for capreomycin to 88.2% (85.1–90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1–92.5%) for moxifloxacin to 99.5% (99.0–99.8%) for amikacin. This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis

The occurrence and dissemination of methicillin and vancomycin-resistant Staphylococcus in samples from patients and health professionals of a university hospital in Recife, State of Pernambuco, Brazil

Introduction Methicillin-resistant Staphylococcus aureus (MRSA) strains have been responsible for many nosocomial outbreaks. Within hospitals, colonized employees often act as reservoirs for the spread of this organism. This study collected clinical samples of 91 patients admitted to the intensive care unit (ICU), hemodialysis/nephrology service and surgical clinic, and biological samples from the nasal cavities of 120 professionals working in those environments, of a University Hospital in Recife, in the State of Pernambuco, Brazil. The main objective of this study was to determine the occurrence and dissemination of methicillin- and vancomycin-resistant Staphylococcus spp. Methods The isolates obtained were tested for susceptibility to oxacillin and vancomycin and detection of the mecA gene. In addition, the isolates were evaluated for the presence of clones by ribotyping-polymerase chain reaction (PCR). Results MRSA occurrence, as detected by the presence of the mecA gene, was more prevalent among nursing technicians; 48.1% (13/27) and 40.7% (11/27) of the isolates were from health professionals of the surgical clinic. In patients, the most frequent occurrence of mecA-positive isolates was among the samples from catheter tips (33.3%; 3/9), obtained mostly from the hemodialysis/nephrology service. Eight vancomycin-resistant strains were found among the MRSA isolates through vancomycin screening. Based on the amplification patterns, 17 ribotypes were identified, with some distributed between patients and professionals. Conclusions Despite the great diversity of clones, which makes it difficult to trace the source of the infection, knowledge of the molecular and phenotypic profiles of Staphylococcus samples can contribute towards guiding therapeutic approaches in the treatment and control of nosocomial infections

Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing

Background: The World Health Organization recommends drug-susceptibility testing of Mycobacterium tuberculosis complex for all patients with tuberculosis to guide treatment decisions and improve outcomes. Whether DNA sequencing can be used to accurately predict profiles of susceptibility to first-line antituberculosis drugs has not been clear. Methods: We obtained whole-genome sequences and associated phenotypes of resistance or susceptibility to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and pyrazinamide for isolates from 16 countries across six continents. For each isolate, mutations associated with drug resistance and drug susceptibility were identified across nine genes, and individual phenotypes were predicted unless mutations of unknown association were also present. To identify how whole-genome sequencing might direct first-line drug therapy, complete susceptibility profiles were predicted. These profiles were predicted to be susceptible to all four drugs (i.e., pansusceptible) if they were predicted to be susceptible to isoniazid and to the other drugs or if they contained mutations of unknown association in genes that affect susceptibility to the other drugs. We simulated the way in which the negative predictive value changed with the prevalence of drug resistance. Results: A total of 10,209 isolates were analyzed. The largest proportion of phenotypes was predicted for rifampin (9660 [95.4%] of 10,130) and the smallest was predicted for ethambutol (8794 [89.8%] of 9794). Resistance to isoniazid, rifampin, ethambutol, and pyrazinamide was correctly predicted with 97.1%, 97.5%, 94.6%, and 91.3% sensitivity, respectively, and susceptibility to these drugs was correctly predicted with 99.0%, 98.8%, 93.6%, and 96.8% specificity. Of the 7516 isolates with complete phenotypic drug-susceptibility profiles, 5865 (78.0%) had complete genotypic predictions, among which 5250 profiles (89.5%) were correctly predicted. Among the 4037 phenotypic profiles that were predicted to be pansusceptible, 3952 (97.9%) were correctly predicted. Conclusions: Genotypic predictions of the susceptibility of M. tuberculosis to first-line drugs were found to be correlated with phenotypic susceptibility to these drugs. (Funded by the Bill and Melinda Gates Foundation and others.

Quinolone/fluoroquinolone susceptibility in Escherichia coli correlates with human polymicrobial bacteriuria and with in vitro interleukine-8 suppression.

Urinary tract infections (UTIs) are frequently polymicrobial diseases mainly sustained by Escherichia coli in association with other opportunistic pathogens. Cystitis and pyelonephritis are usually accompanied by an inflammatory response, which includes neutrophil recruitment. Uropathogenic E. coli possess the ability to evade host defenses, modulating the innate immune response. The aim of this study was to determine whether particular E. coli strains correlate with polymicrobial bacteriuria and whether escape from the early host defenses and microbial synergy could lead to mixed UTIs. We evaluated 188 E. coli-positive urine samples and assessed the relationships among polymicrobism, neutrophil presence and several traits of E. coli isolates (virulence factors such as hlyA, fimA, papC and their relative products, i.e. hemolysin, type 1 and P fimbriae, and cnf1, their phylogenetic group) and their ability to suppress cytokine response in 5637 bladder epithelial cells. Escherichia coli susceptibility toward quinolones and fluoroquinolones, known to be linked to the pathogenicity of this species, was also considered. We found significant correlations among polymicrobial bacteriuria, absence of pyuria and quinolone/fluoroquinolone susceptibility of E. coli isolates and their enhanced capability to suppress interleukin-8 urothelial production when compared with the patterns induced by the resistant strains

Effects of demethylfruticuline A and fruticuline A from Salvia corrugata Vahl. on biofilm production in vitro by multiresistant strains of Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis.

In this study, demethylfruticuline A (dfA) and fruticuline A (fA), two quinones representing the major diterpenoid components of the exudate produced by the aerial parts of Salvia corrugata, were assessed for their ability to modify surface characteristics, such as hydrophobicity, and to inhibit synthesis of biofilm in vitro by multiresistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Five strains of S. aureus (three meticillin-resistant and two meticillin-susceptible), five strains of S. epidermidis (four meticillin-resistant and one meticillin-susceptible) and eight vancomycin-resistant E. faecalis, all recently isolated from clinical specimens and capable of slime production, were studied. fA decrease by at least two-fold the hydrophobic properties of the S. aureus cell membrane but did not affect S. epidermidis or E. faecalis. Biofilm formation on polystyrene plates was quantified spectrophotometrically by established methodologies. Inhibition of biofilm formation was also confirmed by the Congo red agar plate assay. dfA and fA were more effective against S. aureus strains (>70% effect at subinhibitory concentrations) than against S. epidermidis in inhibiting slime synthesis. Against E. faecalis, dfA at subinhibitory concentration induced an inhibition of biofilm production of ca. 60%; fA was less active and more strain-dependent. Moreover, the two compounds were shown to possess chelating activity on divalent and trivalent metal cations. Interactions of fA and dfA with bacteria could be very complex, possibly being species-specific, and could depend not only on inhibition of exopolysaccharide synthesis but also on their chelating activity and on changes in the microorganism's surface, including cell hydrophobicity

New clerodane diterpenoids from Salvia buchananii Hedge

S. buchananii Hedge (Lamiaceae) (subgenus Calosphace) is a Mexican perennial herbaceous species with almost glabrous petiolate and spathulate leaves and large corollas. The antimicrobial activity of the surface exudate obtained by rinsing the aerial parts of with CH2Cl2 was preliminary evaluated by testing 10 \ub5l of 2-fold dilutions (ranging from 1000 mg/L to 64 mg/L) on selected multi-drug-resistant Gram-positive strains, appropriately seeded on agar plates. A clear halo of inhibition was observed on Staphylococcus epidermidis, Staphylococcus aureus (minimum active concentrations for both strains 125 mg/L) and, particularly, on Enterococcus faecium and Enterococcus faecalis (minimum active concentrations for both strains 64 mg/L). The exudate extract, subjected to repeated column chromatography on Sephadex LH-20 and silica gel and to semi-preparative RP-HPLC, yielded two new clerodane diterpenoids, identified by IR and NMR analysis, including TOCSY, COSY, HSQC, HMBC and ROESY experiments, and ESI-TRAP-MS and HR-MS analysis

Antifungal resistance in Candida spp. isolated in Italy between 2002 and 2005 from children and adults

The activity of amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole was tested in vitro against 618 clinical Candida spp. isolates, using the broth microdilution or the disk diffusion method (voriconazole). Amphotericin B and voriconazole were the most potent antifungal agents assayed (100% of susceptible strains). Resistance to fluconazole and itraconazole was detected in three (0.7%) and 11 (2.7%) isolates of Candida albicans and in four (3.7%) isolates of Candida glabrata. Flucytosine intermediate, resistant strains, or both, were observed in C. albicans (0.3% and 0.7%), C. glabrata (2.8% intermediate) and C. tropicalis (15.2% and 15.2%). C. krusei was the least susceptible species to azoles. No statistically significant differences in the rates of resistant isolates depending on site of infection and age of the patient were observed, with the exception of C. albicans and itraconazole (higher percentage of resistance in children). At present, acquired antifungal resistance represents an uncommon finding in most Candida spp. circulating in Northern Italy