Quinolones modulate ghrelin receptor signaling: potential for a novel small molecule scaffold in the treatment of cachexia

Cachexia is a metabolic wasting disorder characterized by progressive weight loss, muscle atrophy, fatigue, weakness, and appetite loss. Cachexia is associated with almost all major chronic illnesses including cancer, heart failure, obstructive pulmonary disease, and kidney disease and significantly impedes treatment outcome and therapy tolerance, reducing physical function and increasing mortality. Current cachexia treatments are limited and new pharmacological strategies are needed. Agonists for the growth hormone secretagogue (GHS-R1a), or ghrelin receptor, prospectively regulate the central regulation of appetite and growth hormone secretion, and therefore have tremendous potential as cachexia therapeutics. Non-peptide GHS-R1a agonists are of particular interest, especially given the high gastrointestinal degradation of peptide-based structures, including that of the endogenous ligand, ghrelin, which has a half-life of only 30 min. However, few compounds have been reported in the literature as non-peptide GHS-R1a agonists. In this paper, we investigate the in vitro potential of quinolone compounds to modulate the GHS-R1a in both transfected human cells and mouse hypothalamic cells. These chemically synthesized compounds demonstrate a promising potential as GHS-R1a agonists, shown by an increased intracellular calcium influx. Further studies are now warranted to substantiate and exploit the potential of these novel quinolone-based compounds as orexigenic therapeutics in conditions of cachexia and other metabolic and eating disorders.Irish Research Council for Science and Technology (IRCSET)Science Foundation Ireland (SFI/12/IP/1315)Science Foundation Ireland (SFI/12/RC/2275)Science Foundation Ireland (SFI/12/RC/2273)Universidad de Sevill

Primer relevamiento de marcadores de resistencia a antibióticos en Enterobacteriaceae en Cochabamba, Bolivia

Se llevó a cabo un relevamiento molecular de la resistencia a antibióticos de importancia clínica en aislamientos recuperados en Cochabamba, Bolivia. Se estudiaron los genes codificantes de β-lactamasas de espectro extendido y de resistencia a quinolonas de localización plasmídica (PMQR) en un total de 101 aislamientos de enterobacterias resistentes a oximinocefalosporinas recuperados en distintos centros de salud, durante 4 meses (2012-2013). En todos ellos se detectó la presencia de cefotaximasas, las CTX-M grupo 1 fueron las más prevalentes (88,1%). La presencia de blaOXA-1 se detectó en el 76,4% de estos aislamientos. Se observó una elevada proporción de aislamientos resistentes a quinolonas. El gen aac(6′)-Ib-cr fue el determinante PMQR más frecuentemente identificado (83%). Además, 6 aislamientos resultaron ser portadores de qnrB. Por otro lado, cabe remarcar que 7 Escherichia coli presentaron qepA1 (6) y oqxAB (1); se documenta así por primera vez la presencia de oqxAB en Bolivia. Este estudio constituye el primer relevamiento de marcadores de resistencia en aislamientos clínicos de enterobacterias en Cochabamba, Bolivia; de este modo se contribuye al conocimiento regional de la situación epidemiológica, la cual presenta un escenario similar al observado en el resto de Latinoamérica.A molecular survey was conducted in Cochabamba, Bolivia, to characterize the mechanism involved in the resistance to clinically relevant antibiotics. Extended Spectrum β-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) markers were investigated in a total of 101 oxyimino-cephalosporin-resistant enterobacteria recovered from different health centers during four months (2012?2013). CTX-M enzymes were detected in all isolates, being the CTX-M-1 group the most prevalent (88.1%). The presence of blaOXA-1 was detected in 76.4% of these isolates. A high quinolone resistance rate was observed among the included isolates. The aac(6′)-Ib-cr gene was the most frequent PMQR identified (83.0%). Furthermore, 6 isolates harbored the qnrB gene. Interestingly, qepA1 (6) and oqxAB (1), were detected in 7 Escherichia coli, being the latter the first to be reported in Bolivia. This study constitutes the first molecular survey on resistance markers in clinical enterobacterial isolates in Cochabamba, Bolivia, contributing to the regional knowledge of the epidemiological situation. The molecular epidemiology observed herein resembles the scene reported in South America.Fil: Saba Villarroel, Paola M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (-1 position). Mutagenesis of -1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable -1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the -1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage

Enzymatic one-step ring contraction for quinolone biosynthesis.

The 6,6-quinolone scaffolds on which viridicatin-type fungal alkaloids are built are frequently found in metabolites that display useful biological activities. Here we report in vitro and computational analyses leading to the discovery of a hemocyanin-like protein AsqI from the Aspergillus nidulans aspoquinolone biosynthetic pathway that forms viridicatins via a conversion of the cyclopenin-type 6,7-bicyclic system into the viridicatin-type 6,6-bicyclic core through elimination of carbon dioxide and methylamine through methyl isocyanate

Mutations that Separate the Functions of the Proofreading Subunit of the Escherichia coli Replicase

The dnaQ gene of Escherichia coli encodes the Ɛ subunit of DNA polymerase III, which provides the 3\u27 - 5\u27 exonuclease proofreading activity of the replicative polymerase. Prior studies have shown that loss of Ɛ leads to high mutation frequency, partially constitutive SOS, and poor growth. In addition, a previous study from our laboratory identified dnaQ knockout mutants in a screen for mutants specifically defective in the SOS response after quinolone (nalidixic acid) treatment. To explain these results, we propose a model whereby, in addition to proofreading, Ɛ plays a distinct role in replisome disassembly and/or processing of stalled replication forks. To explore this model, we generated a pentapeptide insertion mutant library of the dnaQgene, along with site-directed mutants, and screened for separation of function mutants. We report the identification of separation of function mutants from this screen, showing that proofreading function can be uncoupled from SOS phenotypes (partially constitutive SOS and the nalidixic acid SOS defect). Surprisingly, the two SOS phenotypes also appear to be separable from each other. These findings support the hypothesis that Ɛ has additional roles aside from proofreading. Identification of these mutants, especially those with normal proofreading but SOS phenotype(s), also facilitates the study of the role of e in SOS processes without the confounding results of high mutator activity associated with dnaQ knockout mutants

Exercise-induced tendon and bone injury in recreational runners: A test-retest reliability study

Background: Long-distance runners are prone to injuries including Achilles tendinopathy and medial tibial stress syndrome. We have developed an Internet comprehensive self-report questionnaire examining the medical history, injury history, and running habits of adult recreational runners. Objective: The objective of the study was to evaluate two alternative forms of test-retest reliability of a comprehensive self-report Internet questionnaire retrospectively examining the medical history, injury history, and running habits among a sample of adult recreational runners. This will contribute to the broad aims of a wider study investigating genetics and running injury. Methods: Invitations to complete an Internet questionnaire were sent by email to a convenience pilot population (test group 1). Inclusion criteria required participants to be a recreational runner age 18 or over, who ran over 15 km per week on a consistent basis. The survey questions addressed regular running habits and any injuries (including signs, symptoms, and diagnosis) of the lower limbs that resulted in discontinuation of running for a period of 2 consecutive weeks or more, within the last 2 years. Questions also addressed general health, age, sex, height, weight, and ethnic background. Participants were then asked to repeat the survey using the Internet platform again after 10-14 days. Following analysis of test group 1, we soft-launched the survey to a larger population (test group 2), through a local running club of 900 members via email platform. The same inclusion criteria applied, however, participants were asked to complete a repeat of the survey by telephone interview after 7-10 days. Selected key questions, important to clarify inclusion or exclusion from the wider genetics study, were selected to evaluate test-retest reliability. Reliability was quantified using the kappa coefficient for categorical data. Results: In response to the invitation, 28 participants accessed the survey from test group 1, 23 completed the Internet survey on the first occasion, and 20 completed the Internet retest within 10-21 days. Test-retest reliability scored moderate to almost perfect (kappa=.41 to .99) for 19/19 of the key questions analyzed. Following the invitation, 122 participants accessed the survey from test group 2, 101 completed the Internet survey on the first occasion, and 50 were randomly selected and contacted by email inviting them to repeat the survey by telephone interview. There were 33 participants that consented to the telephone interview and 30 completed the questionnaire within 7-10 days. Test-retest reliability scored moderate to almost perfect for 18/19 (kappa=.41 to .99) and slight for 1/19 of the key questions analyzed. Conclusions: We successfully developed a self-reported, retrospective questionnaire, delivered using Internet software, providing stable and reliable answers. We demonstrate that our survey provides a relatively quick, easy to complete, and cost effective method to collect epidemiological data from recreational runners and evaluate these participants for inclusion into a genetic study

The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa

Certain members of the fluorescent pseudomonads produce and secrete phenazines. These heterocyclic, redox-active compounds are toxic to competing organisms, and the cause of these antibiotic effects has been the focus of intense research efforts. It is largely unknown, however, how pseudomonads themselves respond to – and survive in the presence of – these compounds. Using Pseudomonas aeruginosa DNA microarrays and quantitative RT-PCR, we demonstrate that the phenazine pyocyanin elicits the upregulation of genes/operons that function in transport [such as the resistance-nodulation-cell division (RND) efflux pump MexGHI-OpmD] and possibly in redox control (such as PA2274, a putative flavin-dependant monooxygenase), and downregulates genes involved in ferric iron acquisition. Strikingly, mexGHI-opmD and PA2274 were previously shown to be regulated by the PA14 quorum sensing network that controls the production of virulence factors (including phenazines). Through mutational analysis, we show that pyocyanin is the physiological signal for the upregulation of these quorum sensing-controlled genes during stationary phase and that the response is mediated by the transcription factor SoxR. Our results implicate phenazines as signalling molecules in both P. aeruginosa PA14 and PAO1

Effect of pH on ciprofloxacin ozonation in hospital WWTP effluent

A bubble column was used for ozonation of the quinolone antibiotic ciprofloxacin and the effect of pH was tested. Degradation at pH 7 increased the ciprofloxacin half life time to 29.1 min compared to pH 3 (26.8 min) and pH 10 (18.7 min), possibly due to increased sorption at neutral pH. Degradation product identification revealed strongest degradation at the piperazinyl substituent at pH 10 while degradation at the quinolone moiety seems promising at pH 7. For P. fluorescens and E. coli, reduction in antibacterial activity, monitored by agar diffusion tests, was in correlation with the ciprofloxacin degradation rate. For B. coagulans, however, no differences in residual antibacterial activity were found in function of pH, indicating that degradation products also affect antibacterial activity of ozonated samples