Antibiotics do not induce expression of acrAB directly but via a RamA-dependent pathway.

The aim of this study was to determine if acrAB induction in Salmonella Typhimurium relies solely on RamA or if other transcriptional activator pathways are also involved, and to better understand the kinetics of induction of both acrAB and ramA. We evaluated the expression of acrAB in S. Typhimurium in response to a variety of compounds that are known to induce the expression of one or more of the transcriptional activators, MarA, SoxS, RamA, and Rob. We utilized green fluorescent protein (GFP) transcriptional reporter fusions to investigate the changes in the expression of acrAB, ramA, marA, and soxS following exposure to sub-inhibitory concentrations of antimicrobial compounds. Of the compounds tested, 13 induce acrAB expression in S. Typhimurium via RamA, MarA, SoxS, and Rob-dependent pathways. None of the tested antibiotics induced acrAB expression, and compounds that induced acrAB expression also induced a general stress response. The results from this study show that the majority of compounds tested induced acrAB via the RamA-dependent pathway. However, none of the antibiotic substrates of the AcrB efflux pump directly increased the expression of AcrAB either directly or indirectly via the induction of one of the transcriptional activators. Using a dual GFP/RFP reporter, we investigated the kinetics of the induction of ramA and acrAB simultaneously and found that acrAB gene expression was transient compared to ramA gene expression. ramA gene expression increased with time and would remain high or decrease slowly over the course of the experiment indicating that RamA exerts a wider global effect and is not limited to efflux regulation alone.

Investigating transportation research based on social media analysis: a systematic mapping review.

Social media is a pool of users' thoughts, opinions, surrounding environment, situation and others. This pool can be used as a real-time and feedback data source for many domains such as transportation. It can be used to get instant feedback from commuters; their opinions toward the transportation network and their complaints, in addition to the traffic situation, road conditions, events detection and many others. The problem is in how to utilize social media data to achieve one or more of these targets. A systematic review was conducted in the field of transportation-related research based on social media analysis (TRR-SMA) from the years between 2008 and 2018; 74 papers were identified from an initial set of 703 papers extracted from 4 digital libraries. This review will structure the field and give an overview based on the following grounds: activity, keywords, approaches, social media data and platforms and focus of the researches. It will show the trend in the research subjects by countries, in addition to the activity trends, platforms usage trend and others. Further analysis of the most employed approach (Lexicons) and data (text) will be also shown. Finally, challenges and future works are drawn and proposed. Supplementary Information: The online version contains supplementary material available at 10.1007/s11192-021-04046-2.

New Multidrug Efflux Inhibitors for Gram-Negative Bacteria.

Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.

Phosphorylation of conserved phosphoinositide binding pocket regulates sorting nexin membrane targeting.

Sorting nexins anchor trafficking machines to membranes by binding phospholipids. The paradigm of the superfamily is sorting nexin 3 (SNX3), which localizes to early endosomes by recognizing phosphatidylinositol 3-phosphate (PI3P) to initiate retromer-mediated segregation of cargoes to the trans-Golgi network (TGN). Here we report the solution structure of full length human SNX3, and show that PI3P recognition is accompanied by bilayer insertion of a proximal loop in its extended Phox homology (PX) domain. Phosphoinositide (PIP) binding is completely blocked by cancer-linked phosphorylation of a conserved serine beside the stereospecific PI3P pocket. This "PIP-stop" releases endosomal SNX3 to the cytosol, and reveals how protein kinases control membrane assemblies. It constitutes a widespread regulatory element found across the PX superfamily and throughout evolution including of fungi and plants. This illuminates the mechanism of a biological switch whereby structured PIP sites are phosphorylated to liberate protein machines from organelle surfaces.

Structural basis of dynamic membrane recognition by trans-Golgi network specific FAPP proteins.

Glycosphingolipid metabolism relies on selective recruitment of the pleckstrin homology (PH) domains of FAPP proteins to the trans-Golgi network. The mechanism involved is unclear but requires recognition of phosphatidylinositol-4-phosphate (PI4P) within the Golgi membrane. We investigated the molecular basis of FAPP1-PH domain interactions with PI4P bilayers in liposome sedimentation and membrane partitioning assays. Our data reveals a mechanism in which FAPP-PH proteins preferentially target PI4P-containing liquid disordered membranes, while liquid ordered membranes were disfavored. Additionally, NMR spectroscopy was used to identify the binding determinants responsible for recognizing trans-Golgi network-like bicelles including phosphoinositide and neighboring lipid molecules. Membrane penetration by the FAPP1-PH domain was mediated by an exposed, conserved hydrophobic wedge next to the PI4P recognition site and ringed by a network of complementary polar residues and basic charges. Our data illuminates how insertion of a structured loop provides selectivity for sensing membrane fluidity and targeting to defined membrane zones and organelles. The determinants of this membrane sensing process are conserved across the CERT, OSBP and FAPP family. Hence, lipid gradients not only result in differential membrane ordering along the secretory pathway but also specifically localize diverse proteins through recognition of ensembles of lipid ligands in dynamic and deformable bilayers in order to promote anterograde trafficking.

Structural insights into the activation of the RhoA GTPase by the lymphoid blast crisis (Lbc) oncoprotein.

The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.

Cognitive remediation for individuals with psychosis in a supported education setting: a randomized controlled trial.

Cognitive remediation (CR) has demonstrated good outcomes when paired with supported employment, however little is known about its effectiveness when integrated into a supported education program. This randomized controlled trial examined the effectiveness of integrating CR within a supported education program compared with supported education without CR. Thirty-seven students with psychosis were recruited into the study in the 2012 academic year. Academic functioning, cognition, self-esteem, and symptomatology were assessed at baseline, at 4months following the first academic term in which CR was provided, and at 8months assessing maintenance of gains. The treatment group demonstrated better retention in the academic program and a trend of improvement across a range of academic functional domains. While both treatment and control groups showed improvement in cognitive measures, the outcomes were not augmented by CR training. CR was also associated with significant and sustained improvements in self esteem. Further research, investigating specific intervention components is required to clarify the mixed findings regarding the effectiveness of CR in an education setting.