Detection of vancomycin variable enterococci (VVE) among clinical isolates of Enterococcus faecium collected across India-first report from the subcontinent

PurposeEmergence of vancomycin variable enterococci (VVE) poses a challenge to empiric vancomycin therapy. Vancomycin-variable enterococci (VVE) are vanA-positive, yet phenotypically vancomycin-susceptible enterococci that can switch to a vancomycin-resistant phenotype when exposed to vancomycin. The aim of the present study was to determine the prevalence of VVE in India.MethodsIsolates of phenotypically vancomycin susceptible Enterococcus faecium from 20 tertiary care hospitals across India were collected and tested for the presence of vanA, vanR, vanS, vanB and vanC genes by conventional PCR using previously published primers. Isolates positive for vanA gene were considered as VVE.ResultsThe prevalence of VVE was 1.5% (5/340). Only one VVE isolate was positive for vanR and vanS, and all the isolates were negative for vanB and vanC.ConclusionsAlthough the prevalence is low, our finding emphasizes the importance of routinely screening for van genes in enterococci that are phenotypically susceptible. Silenced vanA able to escape detection and revert to resistance during vancomycin therapy represents a new challenge in clinical settings.     

Tangeretin ameliorates oxidative stress in the renal tissues of rats with experimental breast cancer induced by 7,12-dimethylbenz[a]anthracene

Tangeretin, a citrus polymethoxyflavone, is an antioxidant modulator which has been shown to exhibit a surfeit of pharmacological properties. The present study was hypothesized to explore the therapeutic activity of tangeretin against 7,12-dimethylbenz[a]anthracene (DMBA) induced kidney injury in mammary tumor bearing rats. Recently, we have reported the chemotherapeutic effect of tangeretin in the breast tissue of DMBA induced rats. Breast cancer was induced by “air pouch technique” with a single dose of 25 mg/kg of DMBA. Tangeretin (50 mg/kg/day) was administered orally for four weeks. The renoprotective nature of tangeretin was assessed by analyzing the markers of oxidative stress, proinflammatory cytokines and antioxidant competence in DMBA induced rats. Tangeretin treatment revealed a significant decline in the levels of lipid peroxides, inflammatory cytokines and markers of DNA damage, and a significant improvement in the levels of enzymatic and non-enzymatic antioxidants in the kidney tissue. Similarly, mRNA, protein and immunohistochemical analysis substantiated that tangeretin treatment notably normalizes the renal expression of Nrf2/Keap1, its downstream regulatory proteins and the inflammatory cytokines in the DMBA induced rats. Histological and ultrastructural observations also evidenced that the treatment with tangeretin effectively protects the kidney from DMBA-mediated oxidative damage, hence, proving its nephroprotective nature.     

Notch-activated signaling cascade interacts with mitochondrial remodeling proteins to regulate cell survival

Survival of differentiated cells is one of several processes regulated by Notch activity, although the general principles underlying this function remain to be characterized. Here, we probe the mechanism underlying Notch-mediated survival, building on emerging evidence that apoptotic responses coordinated by specialized intermediates converge on mitochondria, identifying a core event in death pathways. The Bcl-2 family protein Bax is one such intermediate, which in a unifying response to diverse apoptotic stimuli nucleates multiprotein assemblies on mitochondria, committing cells to irrevocable damage. Using Bax as the prototype stimulus, we analyze Notch signaling for potential interactions with mitochondria, probe intrinsic properties of the Notch receptor, and describe key intermediates in the Notch-activated signaling cascade. Ligand-dependent processing was necessary to generate the Notch intracellular domain (NIC) although signaling was independent of canonical interactions with nuclear factors. Notably, antiapoptotic activity was recapitulated by NIC recombinants, localized outside the nucleus, and compromised by enforced nuclear sequestration. NIC signaled via the kinase Akt to prevent the loss of mitochondrial function, contiguity, and consequent nuclear damage, outcomes critically depend on mitochondrial remodeling proteins Mitofusins-(Mfn)-1 and 2. Thus, the NIC-Akt-Mfn signaling cascade identifies a pathway regulating cell-survival, independent of canonical functions associated with NIC activity.     

Evaluation of beta defensin 2 production by chicken heterophils using direct MALDI mass spectrometry

Beta defensins (BD) are cysteine rich, cationic antimicrobial peptides (AMP) produced mainly by epithelial and myeloid cells such as neutrophils. In birds, the neutrophil equivalent heterophils produce avian beta defensins (AvBD) of which AvBD2 is the major isoform. Heterophils recognize pathogens or their derived products through a series of pattern recognition receptors called toll-like receptors (TLR) leading to their antimicrobial activities. This work is the first report of TLR modulation of AvBD2 expression in chickens. To measure the effect of TLR activation on AvBD2 production, the heterophils were cultured with different TLR agonists for 6 h. Modulation of AvBD2 levels by TLR activation was measured using direct MALDI mass spectrometry without stable isotopic labeling or chromatographic separation. Chemical modification of the conditioned media was performed using reduction/alkylation with dithiothreitol/iodoacetamide to distinguish TLR treated AvBD2 (reduced/alkylated) from controls (non-reduced). Changes in corrected ion intensity ratios were assumed to reflect AvBD2 modulation in heterophils upon activation with different TLR agonists. In general, TLR agonists increased AvBD2 production with LPS showing the greatest induction and CpG-ODN showing little or no effect. These data show that the direct MALDI-MS coupled with reduction/alkylation may provide a rapid relative quantitative approach to the measurement of agonist-induced differential expression of AvBD2.     

The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers.

Several epidemiologic studies indicate that NAT2-related slow N-acetylation increases bladder cancer risk among workers exposed to aromatic amines, presumably because N-acetylation is important for the detoxification of these compounds. Previously, we showed that NAT2 polymorphisms did not influence bladder cancer risk among Chinese workers exposed exclusively to benzidine (BZ), suggesting that NAT2 N-acetylation is not a critical detoxifying pathway for this aromatic amine. To evaluate the biologic plausibility of this finding, we carried out a cross-sectional study of 33 workers exposed to BZ and 15 unexposed controls in Ahmedabad, India, to evaluate the presence of BZ-related DNA adducts in exfoliated urothelial cells, the excretion pattern of BZ metabolites, and the impact of NAT2 activity on these outcomes. Four DNA adducts were significantly elevated in exposed workers compared to controls; of these, the predominant adduct cochromatographed with a synthetic N-(3'- phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine standard and was the only adduct that was significantly associated with total BZ urinary metabolites (r = 0.68, P < 0.0001). To our knowledge this is the first report to show that BZ forms DNA adducts in exfoliated urothelial cells of exposed humans and that the predominant adduct formed is N-acetylated, supporting the concept that monofunctional acetylation is an activation, rather than a detoxification, step for BZ. However, because almost all BZ-related metabolites measured in the urine of exposed workers were acetylated among slow, as well as rapid, acetylators (mean +/- SD 95 +/- 1.9% vs. 97 +/- 1.6%, respectively) and NAT2 activity did not affect the levels of any DNA adduct measured, it is unlikely that interindividual variation in NAT2 function is relevant for BZ-associated bladder carcinogenesis.     

How Do Hospitalized Patients Feel About Resident Work Hours,Fatigue, and Discontinuity of Care?

BACKGROUND: Patient-centered care requires that physicians understand patients' perspectives. Since the resident work hour rules were instituted, little information is available about how patients perceive these issues. Our objectives were to explore patients' knowledge, concerns, and attitudes about resident work hours, fatigue, and continuity of inpatient care and to evaluate the association between patients' trust and satisfaction with these concerns and attitudes. METHODS: We conducted a cross-sectional survey of 134 internal medicine inpatients at 3 institutions including a tertiary care academic health center, a Veterans Affairs medical center, and a private community teaching hospital. RESULTS: Mean age was 59 (range, 24-90), with 60% men and 70% white. Most patients agreed (50%) or felt neutral (38%) toward resident work hours being limited. Patients estimated that residents worked 60 h per week but thought that they should work no more than 51 h per week (p < .01 for the difference). Twenty-seven percent of patients had some concern about fatigue in the residents, and 28% reported concern about how often hand-offs of care occurred. Factor analysis yielded 3 factors: "worried about discontinuity/fatigue," "attitude toward resident/nurse work hours," and "perceived resident/nurse fatigue." In multivariable analyses, the "worried about fatigue/discontinuity" factor significantly predicted trust and satisfaction, and the "perceived resident/nurse fatigue" factor also predicted satisfaction. CONCLUSIONS: Some inpatients are concerned about both fatigue in resident physicians and discontinuity of care. This may play a role in trust and satisfaction for patients. Taking steps to design systems to minimize fatigue and discontinuity would be ideal.