A novel bacterial l-arginine sensor controlling c-di-GMP levels in Pseudomonas aeruginosa

Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host–pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signaling molecules able to influence group behavior in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3′, 5′-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an l-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as l-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first l-arginine sensor able to link environment sensing, c-di-GMP signaling and biofilm formation in this bacterium

Creating and Managing Dynamic Cloud Federations

Cloud computing has evolved from a promising approach to the service provisioning to the reference model for all new data centres to build. Additionally, an increasing number of companies are choosing to migrate their business in the cloud "ecosystem" adopting the solutions developed by the biggest public Cloud Service Providers (CSPs). Smaller CSPs build their infrastructure on technologies available and to better support user activities and provide enough resources to their users, the federation could be a possible solution. In this work, we present different federation models, showing their strengths and weakness together with our considerations. Beside the highlighted existing federation we show the design of a new implementations under development at INFN aiming at maximising the scalability and flexibility of small and/or hybrid clouds by the introduction of a federation manager. This new component will support a seamless resources renting on the base of acceptance of federation agreements among operators. Additionally, we will discuss how the implementation of this model inside research institutes could help in the field of High Energy Physics with explicit reference at LHC experiments, digital humanities, life sciences and others

Studying ggdef domain in the act: Minimize conformational frustration to prevent artefacts

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies

Targeting the Interaction between the SH3 Domain of Grb2 and Gab2

Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction

The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism

Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5'untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1-UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism

A 2-Year Pragmatic Trial of Antibiotic Stewardship in 27 Community Nursing Homes

OBJECTIVES: To determine if antibiotic prescribing in community nursing homes (NHs) can be reduced by a multicomponent antibiotic stewardship intervention implemented by medical providers and nursing staff and whether implementation is more effective if performed by a NH chain or a medical provider group. DESIGN: Two-year quality improvement pragmatic implementation trial with two arms (NH chain and medical provider group). SETTING: A total of 27 community NHs in North Carolina that are typical of NHs statewide, conducted before announcement of the US Centers for Medicare and Medicaid Services antibiotic stewardship mandate. PARTICIPANTS: Nursing staff and medical care providers in the participating NHs. INTERVENTION: Standardized antibiotic stewardship quality improvement program, including training modules for nurses and medical providers, posters, algorithms, communication guidelines, quarterly information briefs, an annual quality improvement report, an informational brochure for residents and families, and free continuing education credit. MEASUREMENTS: Antibiotic prescribing rates per 1000 resident days overall and by infection type; rate of urine test ordering; and incidence of Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA) infections. RESULTS: Systemic antibiotic prescription rates decreased from baseline by 18% at 12 months (incident rate ratio [IRR] = 0.82; 95% confidence interval [CI] = 0.69-0.98) and 23% at 24 months (IRR = 0.77; 95% CI = 0.65-0.90). A 10% increase in the proportion of residents with the medical director as primary physician was associated with a 4% reduction in prescribing (IRR = 0.96; 95% CI = 0.92-0.99). Incidence of C. difficile and MRSA infections, hospitalizations, and hospital readmissions did not change significantly. No adverse events from antibiotic nonprescription were reported. Estimated 2-year implementation costs per NH, exclusive of medical provider time, ranged from $354 to$3653. CONCLUSIONS: Antibiotic stewardship programs can be successfully disseminated in community NHs through either NH administration or medical provider groups and can achieve significant reductions in antibiotic use for at least 2 years. Medical director involvement is an important element of program success