A model for the study of ligand binding to the ribosomal RNA helix h44.

Oligonucleotide models of ribosomal RNA domains are powerful tools to study the binding and molecular recognition of antibiotics that interfere with bacterial translation. Techniques such as selective chemical modification, fluorescence labeling and mutations are cumbersome for the whole ribosome but readily applicable to model RNAs, which are readily crystallized and often give rise to higher resolution crystal structures suitable for detailed analysis of ligand-RNA interactions. Here, we have investigated the HX RNA construct which contains two adjacent ligand binding regions of helix h44 in 16S ribosomal RNA. High-resolution crystal structure analysis confirmed that the HX RNA is a faithful structural model of the ribosomal target. Solution studies showed that HX RNA carrying a fluorescent 2-aminopurine modification provides a model system that can be used to monitor ligand binding to both the ribosomal decoding site and, through an indirect effect, the hygromycin B interaction region

Results of A Local Combination Therapy Antibiogram For \u3cem\u3ePseudomonas Aeruginosa\u3c/em\u3e Isolates: Is Double Worth The Trouble?

Purpose: To determine the frequency at which fluoroquinolones and aminoglycosides demonstrate in vitro activity against non-urinary, non-skin/skin structure Pseudomonas aeruginosa isolates exhibiting decreased susceptibilities to one or more β-lactam agents. Methods: β-lactam-non-susceptible P. aeruginosa isolates recovered from blood, bone, lower respiratory tract, pleural fluid, cerebrospinal fluid, or peritoneal fluid cultures between October 2010 and October 2014 were reviewed from four community hospitals within a single health-system. Only the first isolate per patient was included for analysis. The likelihood that each isolate was susceptible to a non-β-lactam antimicrobial was then determined and summarized within a combination antibiogram. Results: In total, 179 P. aeruginosa isolates with decreased susceptibilities to one or more β-lactam agents were assessed. Because no appreciable differences in antimicrobial susceptibility profile were observed between hospitals, the isolates were evaluated in aggregate. Susceptibility rates for β-lactam monotherapy ranged from 34% to 75%. Aminoglycosides possessed increased antibacterial activity compared to fluoroquinolones. Tobramycin was the non-β-lactam most likely to expand antimicrobial coverage against β-lactam-non-susceptible P. aeruginosa with activity against 64%, 66%, and 65% of cefepime-, piperacillin-tazobactam-, and meropenem-non-susceptible isolates, respectively (p \u3c 0.001 for all). Conclusions: The results of this study support the use of aminoglycosides over fluoroquinolones for achieving optimal, empiric antimicrobial combination therapy for P. aeruginosa when dual antimicrobial therapy is clinically necessary. Future efforts aimed at optimizing combination therapy for P. aeruginosa should focus on systemic interventions that limit the selection of fluoroquinolones in combination with β-lactams to expand coverage based on local susceptibility rates

Host metabolites stimulate the bacterial proton motive force to enhance the activity of aminoglycoside antibiotics

<div><p>Antibiotic susceptibility of bacterial pathogens is typically evaluated using <i>in vitro</i> assays that do not consider the complex host microenvironment. This may help explaining a significant discrepancy between antibiotic efficacy <i>in vitro</i> and <i>in vivo</i>, with some antibiotics being effective <i>in vitro</i> but not <i>in vivo</i> or vice versa. Nevertheless, it is well-known that antibiotic susceptibility of bacteria is driven by environmental factors. Lung epithelial cells enhance the activity of aminoglycoside antibiotics against the opportunistic pathogen <i>Pseudomonas aeruginosa</i>, yet the mechanism behind is unknown. The present study addresses this gap and provides mechanistic understanding on how lung epithelial cells stimulate aminoglycoside activity. To investigate the influence of the local host microenvironment on antibiotic activity, an <i>in vivo</i>-like three-dimensional (3-D) lung epithelial cell model was used. We report that conditioned medium of 3-D lung cells, containing secreted but not cellular components, potentiated the bactericidal activity of aminoglycosides against <i>P</i>. <i>aeruginosa</i>, including resistant clinical isolates, and several other pathogens. In contrast, conditioned medium obtained from the same cell type, but grown as conventional (2-D) monolayers did not influence antibiotic efficacy. We found that 3-D lung cells secreted endogenous metabolites (including succinate and glutamate) that enhanced aminoglycoside activity, and provide evidence that bacterial pyruvate metabolism is linked to the observed potentiation of antimicrobial activity. Biochemical and phenotypic assays indicated that 3-D cell conditioned medium stimulated the proton motive force (PMF), resulting in increased bacterial intracellular pH. The latter stimulated antibiotic uptake, as determined using fluorescently labelled tobramycin in combination with flow cytometry analysis. Our findings reveal a cross-talk between host and bacterial metabolic pathways, that influence downstream activity of antibiotics. Understanding the underlying basis of the discrepancy between the activity of antibiotics <i>in vitro</i> and <i>in vivo</i> may lead to improved diagnostic approaches and pave the way towards novel means to stimulate antibiotic activity.</p></div

Strategies against nonsense: oxadiazoles as translational readthrough-inducing drugs (TRIDs)

This review focuses on the use of oxadiazoles as translational readthrough-inducing drugs (TRIDs) to rescue the functional full-length protein expression in mendelian genetic diseases caused by nonsense mutations. These mutations in specific genes generate premature termination codons (PTCs) responsible for the translation of truncated proteins. After a brief introduction on nonsense mutations and their pathological effects, the features of various classes of TRIDs will be described discussing differences or similarities in their mechanisms of action. Strategies to correct the PTCs will be presented, particularly focusing on a new class of Ataluren-like oxadiazole derivatives in comparison to aminoglycosides. Additionally, recent results on the efficiency of new candidate TRIDs in restoring the production of the cystic fibrosis transmembrane regulator (CFTR) protein will be presented. Finally, a prospectus on complementary strategies to enhance the effect of TRIDs will be illustrated together with a conclusive paragraph about perspectives, opportunities, and caveats in developing small molecules as TRIDs

Diagnosis, evaluation, and management of acute kidney injury : a KDIGO summary (part 1)

Acute kidney injury (AKI) is a common and serious problem affecting millions and causing death and disability for many. In 2012, Kidney Disease: Improving Global Outcomes completed the first ever, international, multidisciplinary, clinical practice guideline for AKI. The guideline is based on evidence review and appraisal, and covers AKI definition, risk assessment, evaluation, prevention, and treatment. In this review we summarize key aspects of the guideline including definition and staging of AKI, as well as evaluation and nondialytic management. Contrast-induced AKI and management of renal replacement therapy will be addressed in a separate review. Treatment recommendations are based on systematic reviews of relevant trials. Appraisal of the quality of the evidence and the strength of recommendations followed the Grading of Recommendations Assessment, Development and Evaluation approach. Limitations of the evidence are discussed and a detailed rationale for each recommendation is provided

Drug-induced stress granule formation protects sensory hair cells in mouse cochlear explants during ototoxicity

Stress granules regulate RNA translation during cellular stress, a mechanism that is generally presumed to be protective, since stress granule dysregulation caused by mutation or ageing is associated with neurodegenerative disease. Here, we investigate whether pharmacological manipulation of the stress granule pathway in the auditory organ, the cochlea, affects the survival of sensory hair cells during aminoglycoside ototoxicity, a common cause of acquired hearing loss. We show that hydroxamate (-)-9, a silvestrol analogue that inhibits eIF4A, induces stress granule formation in both an auditory cell line and ex-vivo cochlear cultures and that it prevents ototoxin-induced hair-cell death. In contrast, preventing stress granule formation using the small molecule inhibitor ISRIB increases hair-cell death. Furthermore, we provide the first evidence of stress granule formation in mammalian hair cells in-vivo triggered by aminoglycoside treatment. Our results demonstrate that pharmacological induction of stress granules enhances cell survival in native-tissue, in a clinically-relevant context. This establishes stress granules as a viable therapeutic target not only for hearing loss but also other neurodegenerative diseases.EI:595 - Action on Hearing Loss; 091092/Z/09/Z - Wellcome Trust (Wellcome); MR/N004329/1 - RCUK | Medical Research Council (MRC)Published versio

Advanced Screencasting With Embedded Assessments in Pathophysiology and Therapeutics Course Modules

Objective. To implement and assess the effectiveness of a hybrid learning model using advanced screencasting with embedded assessments in pathophysiology and therapeutics modules. Design. Two pathophysiology and therapeutics course modules on viral hepatitis and the clinical pharmacokinetics of aminoglycosides were chosen for study. The preclass portion of the hybrid model involved student completion of interactive e-lectures that were created with the use of advanced screencasting and included embedded assessments. Students viewed the e-lectures and completed the assessment questions prior to in-class lecture. Assessment. Preimplementation and postimplementation test scores were compared and student survey data were analyzed. Test scores improved significantly and students’ perceptions of the learning method were favorable. Test scores improved most significantly on higher-level Bloom’s taxonomy questions. Conclusion. A hybrid model that used advanced screencasting with embedded assessments offered a novel method to afford students active-learning opportunities to progress to higher cognitive domains of learning

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

peer-reviewedBifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.Fiona Fouhy is in receipt of an Irish Research Council for Science, Engineering and Technology EMBARK scholarship and is a Teagasc Walsh fellow. Research in the PDC laboratory is supported by the Irish Government under the National Development Plan through the Science Foundation Ireland Investigator award 11/PI/1137. Research in the RPR, CS, PDC and DvS laboratories is also supported by the Science Foundation of Ireland-funded Centre for Science, Engineering and Technology, the Alimentary Pharmabiotic Centre (grant no.s 02/CE/B124 and 07/CE/B1368) and a HRB postdoctoral fellowship (Grant no. PDTM/20011/9) awarded to MOCM