The reaction mechanism of metallo-beta-lactamases is tuned by the conformation of an active site mobile loop

Carbapenems are "last resort" β-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-β-lactamases (MβLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all β-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MβLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MβLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different β-lactams in all MβLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MβLs.Fil: Palacios, Antonela Rocio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Mojica, María F.. Case Western Reserve University; Estados UnidosFil: Giannini, Estefanía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Taracila, Magdalena A.. Case Western Reserve University; Estados Unidos. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Bethel, Christopher R.. Louis Stokes Veterans Affairs Medical Center; Estados UnidosFil: Alzari, Pedro M.. Institut Pasteur de Paris; FranciaFil: Otero, Lisandro Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Klinke, Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llarrull, Leticia Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bonomo, Robert A.. Case Western Reserve University; Estados UnidosFil: Vila, Alejandro Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

A novel role for thrombopoietin in regulating osteoclast development in humans and mice

Emerging data suggest that megakaryocytes (MKs) play a significant role in skeletal homeostasis. Indeed, osteosclerosis observed in several MK-related disorders may be a result of increased numbers of MKs. In support of this idea, we have previously demonstrated that MKs increase osteoblast (OB) proliferation by a direct cell-cell contact mechanism and that MKs also inhibit osteoclast (OC) formation. As MKs and OCs are derived from the same hematopoietic precursor, in these osteoclastogenesis studies we examined the role of the main MK growth factor, thrombopoietin (TPO) on OC formation and bone resorption. Here we show that TPO directly increases OC formation and differentiation in vitro. Specifically, we demonstrate the TPO receptor (c-mpl or CD110) is expressed on cells of the OC lineage, c-mpl is required for TPO to enhance OC formation in vitro, and TPO activates the mitogen-activated protein kinases, Janus kinase/signal transducer and activator of transcription, and nuclear factor-kappaB signaling pathways, but does not activate the PI3K/AKT pathway. Further, we found TPO enhances OC resorption in CD14+CD110+ human OC progenitors derived from peripheral blood mononuclear cells, and further separating OC progenitors based on CD110 expression enriches for mature OC development. The regulation of OCs by TPO highlights a novel therapeutic target for bone loss diseases and may be important to consider in the numerous hematologic disorders associated with alterations in TPO/c-mpl signaling as well as in patients suffering from bone disorders

The Influence Of The Internet On Health Seeking Behaviour Of Nursing Mothers In Ekiti State, Nigeria

The use of internet services to investigate health-related issues is now on the increase among nursing mothers in Nigeria; however, the quality of health information provided on various online sites is questionable. Unfortunately, very little studies have been conduced to explore the influence the internet has on the health seeking behaviour of nursing mothers in Nigeria. This study investigates the influence of the internet on health seeking behaviours of nursing mothers and examines internet‟s influence on their health seeking behaviour. A standardized 10-question survey on Internet use and health seeking behaviour was given to 150 nursing mothers in Ekiti State. It was found that out of 142 responses received, 109 nursing mothers (76.7%) reported using the Internet to find health information. 105 nursing mothers (96.3%) out of the 109 consult the internet for answers to their babies‟ health problems before consulting a doctor or a care giver. 81.6% of the nursing mothers adhere to the online physician‟s advice thereby leading to self medication. An aggregate of 101 (92.6%) nursing mothers submitted that the internet influences their health seeking decisions. Conclusively, the tests of hypothesis show a significant relationship between the use of internet and health seeking behaviours of nursing mothers and also, between the use of internet and self medication among them

Streamlining search methods to update evidence and gap maps: a case study using intergenerational interventions

Background Evidence and Gap Maps (EGMs) should be regularly updated. Running update searches to find new studies for EGMs can be a time-consuming process. Search Summary Tables (SSTs) can help streamline searches by identifying which resources were most lucrative for identifying relevant articles, and which were redundant. The aim of this study was to use an SST to streamline search methods for an EGM of studies about intergenerational activities. Methods To produce the EGM, 15 databases were searched. 8638 records were screened and 500 studies were included in the final EGM. Using an SST, we determined which databases and search methods were the most efficient in terms of sensitivity and specificity for finding the included studies. We also investigated whether any database performed particularly well for returning particular study types. For the best performing databases we analysed the search terms used to streamline the strategies. Results No single database returned all of the studies included in the EGM. Out of 500 studies PsycINFO returned 40% (n = 202), CINAHL 39% (n = 194), Ageline 25% (n = 174), MEDLINE 23% (n = 117), ERIC 20% (n = 100) and Embase 19% (n = 98). HMIC database and Conference Proceedings Citation Index-Science via Web of Science returned no studies that were included in the EGM. ProQuest Dissertations & Theses (PQDT) returned the highest number of unique studies (n = 42), followed by ERIC (n = 33) and Ageline (n = 29). Ageline returned the most randomised controlled trials (42%) followed by CINAHL (34%), MEDLINE (29%) and CENTRAL (29%). CINAHL, Ageline, MEDLINE and PsycINFO performed the best for locating systematic reviews. (62%, 46% and 42% respectively). CINAHL, PsycINFO and Ageline performed best for qualitative studies (41%, 40% and 34%). The Journal of Intergenerational Relationships returned more included studies than any other journal (16%). No combinations of search terms were found to be better in terms of balancing specificity and sensitivity than the original search strategies. However, strategies could be reduced considerably in terms of length without losing key, unique studies. Conclusion Using SSTs we have developed a method for streamlining update searches for an EGM about intergenerational activities. For future updates we recommend that MEDLINE, PsycINFO, ERIC, Ageline, CINAHL and PQDT are searched. These searches should be supplemented by hand-searching the Journal of Intergenerational Relationships and carrying out backwards citation chasing on new systematic reviews. Using SSTs to analyse database efficiency could be a useful method to help streamline search updates for other EGMs

Insights Into the Inhibition of MOX-1 \u3b2-Lactamase by S02030, a Boronic Acid Transition State Inhibitor

The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C \u3b2-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-\uc5 resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 \u3b2-lactamase, a plasmid-borne, expanded-spectrum AmpC \u3b2-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the \u3b2-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 \u3bcg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 \u3bcg/ml when S02030 is added at a concentration of 4 \u3bcg/ml. The IC50 and Ki of S02030 vs. MOX-1 were 1.25 \ub1 0.34 and 0.56 \ub1 0.03 \u3bcM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC \u3b2-lactamases

Blending Geospatial Technology and Traditional Ecological Knowledge to Enhance Restoration Decision-Support Processes in Coastal Louisiana

More informed coastal restoration decisions have become increasingly important given limited resources available for restoration projects and the increasing magnitude of marsh degradation and loss across the Gulf Coast. This research investigated the feasibility and benefits of integrating geospatial technology with the traditional ecological knowledge (TEK) of an indigenous Louisiana coastal population to assess the impacts of current and historical ecosystem change on community viability. The primary goal was to provide coastal resource managers with a decision-support tool that allows for a more comprehensive method of assessing localized ecological change in the Gulf Coast region, which can also benefit human community sustainability. Using remote sensing (RS) and geographic information systems (GIS) mapping products, integrated with a coastal community’s TEK to achieve this goal, the research team determined a method for producing vulnerability/sustainability mapping products for an ecosystem-dependent livelihood base of a coastal population based on information derived from RS imagery prioritized with TEK. This study also demonstrates how such an approach can engage affected community residents who are interested in determining and addressing the causes and mitigating the decline of marsh habitat. Historical image data sets of the study area were acquired to understand evolution of land change to current conditions and project future vulnerability. Image-processing procedures were developed and applied to produce maps that detail land change in the study area at time intervals from 1968 to 2009. This information was combined in a GIS with acquired TEK and scientific data sets relating to marsh vegetation health and vulnerability characteristics to produce mapping products that provide new information for use in the coastal restoration decision-making process. This information includes: (1) marsh areas that are most vulnerable; and (2) the areas that are most significant to community sustainability

Prediction and validation of exenatide risk marker effects on progression of renal disease:Insights from EXSCEL

Aim To assess whether the previously developed multivariable risk prediction framework (PRE score) could predict the renal effects observed in the EXSCEL cardiovascular outcomes trial using short-term changes in cardio-renal risk markers. Materials and Methods Changes from baseline to 6 months in HbA1c, systolic blood pressure (SBP), body mass index (BMI), haemoglobin, total cholesterol, and new micro- or macroalbuminuria were evaluated. The renal outcomes were defined as a composite of a sustained 30% or 40% decline in estimated glomerular filtration rate (eGFR) or end-stage renal disease (ESRD). Relationships between risk markers and long-term renal outcomes were determined in patients with type 2 diabetes from the ALTITUDE study using multivariable Cox regression analysis, and then applied to short-term changes in risk markers observed in EXSCEL to predict the exenatide-induced impact on renal outcomes. Results Compared with placebo, mean HbA1c, BMI, SBP and total cholesterol were lower at 6 months with exenatide, as was the incidence of new microalbuminuria. The PRE score predicted a relative risk reduction for the 30% eGFR decline + ESRD endpoint of 11.3% (HR 0.89; 95% CI 0.83-0.94), compared with 12.7% (HR 0.87; 0.77-0.99) observed risk reduction. For the 40% eGFR decline + ESRD endpoint, the predicted and observed risk reductions were 11.0% (HR 0.89; 0.82-0.97) and 13.7% (HR 0.86, 0.72-1.04), respectively. Conclusions Integrating short-term risk marker changes into a multivariable risk score predicted the magnitude of renal risk reduction observed in EXSCEL

Plasmodium falciparum isolates from southern Ghana exhibit polymorphisms in the SERCA-type PfATPase6 though sensitive to artesunate in vitro

<p>Abstract</p> <p>Background</p> <p>In 2005, Ghana replaced chloroquine with artemisinin-based combination therapy as the first-line treatment for uncomplicated malaria. The aim of this work was to determine for the first time, polymorphisms in the putative <it>pfATPase6 </it>and <it>pftctp</it>, <it>pfmdr1</it>, <it>pfcrt </it>genes in Ghanaian isolates, particularly at a time when there is no report on artemisinin resistance in malaria parasites from Ghana. The sensitivity of parasite isolates to anti-malaria drugs were also evaluated for a possible association with polymorphisms in these genes.</p> <p>Methods</p> <p>The prevalence of point mutations in the above <it>Plasmodium falciparum </it>genes were assessed from filter-paper blood blot samples by DNA sequencing. <it>In vitro </it>drug sensitivity test was carried out on some of the blood samples from volunteers visiting hospitals/clinics in southern Ghana using a modified version of the standard WHO Mark III micro-test.</p> <p>Results</p> <p>All successfully tested parasite isolates were sensitive to artesunate; while 19.4%, 29.0% and 51.6% were resistant to quinine, amodiaquine and chloroquine respectively. The geometric mean of IC₅₀value for artesunate was 0.73 nM (95% CI, 0.38-1.08), amodiaquine 30.69 nM (95% CI, 14.18-47.20) and chloroquine 58.73 nM (95% CI, 38.08-79.38). Twenty point mutations were observed in <it>pfATPase6 </it>gene, with no L263E and S769N. All mutations found were low in frequency, except D639G which was observed in about half of the isolates but was not associated with artesunate response (<it>p </it>= 0.42). The <it>pftctp </it>gene is highly conserved as no mutation was observed, while CVIET which is chloroquine-resistant genotype at codon 72-76 of the <it>pfcrt </it>gene was identified in about half of the isolates; this was consistent with chloroquine IC₅₀values (<it>p </it>= 0.001). Mutations were present in <it>pfmdr1 </it>gene but were not associated with artemisinin response (<it>p </it>= 1.00).</p> <p>Conclusion</p> <p>The <it>pfATPase6 </it>gene is highly polymorphic with D639G appearing to be fixed in Ghanaian isolates. These may just be spontaneous mutations as all parasite isolates that were tested displayed satisfactory <it>in vitro </it>response to artesunate. However, there is no improvement in susceptibility of the parasites to chloroquine five years after its proscription.</p