A new and efficient synthetic route for the anxiolytic agent CL285032

CL285032 is an anxiolytic compound currently under investigation as a possible treatment for canine noise phobia associated anxiety. A robust scale-up and manufacturing process is essential for the development and marketability of the drug. The current synthetic route, although reliable, requires seven steps and has a low overall yield (18%), leaving opportunity for improvement. We are presenting an efficient alternative approach toward the synthesis of CL285032 and the results thereof.     

Helicobacter pylori versus the host: remodeling of the bacterial outer membrane is required for survival in the gastric mucosa

Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4'-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4'-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4'-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4'-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.     

Collaboration in scientific research: a critical need for freshwater ecology

1. Collaboration means actively working together to achieve things which could not be done alone. This article attempts to provide an overall, unified, guiding framework for collaboration in freshwater ecology by discussing aspects of collaboration at individual and organizational levels, and addressing international linkages.
2. The essential elements of collaboration are communication and trust, and effective project management. Barriers to effective collaboration include competition, organizational cultures and organizational instabilities.
3. The success of collaboration can be measured by tangible benefits such as increased numbers of peer-reviewed publications, the production of working models and a number of intangible benefits.
4. Interactions between individuals lie at the heart of an effective collaboration; organizational arrangements should facilitate this interaction. Some governments are encouraging collaboration to increase cost efficiency and allocate accountability. This trend should continue on an international level.
5. Collaboration is a key to future research in freshwater ecology.     

Genome-wide screening for gene function using RNAi in mammalian cells

Mammalian genome sequencing has identified numerous genes requiring functional annotation. The discovery that dsRNA can direct gene-specific silencing in both model organisms and mammalian cells through RNA interference (RNAi) has provided a platform for dissecting the function of independent genes. The generation of large-scale RNAi libraries targeting all predicted genes within mouse, rat and human cells, combined with the large number of cell-based assays, provides a unique opportunity to perform high-throughput genetics in these complex cell systems. Many different formats exist for the generation of genome-wide RNAi libraries for use in mammalian cells. Furthermore, the use of these libraries in either genetic screens or genetic selections allows for the identification of known and novel genes involved in complex cellular phenotypes and biological processes, some of which underpin human disease. In this review, we examine genome-wide RNAi libraries used in model organisms and mammalian cells and provide examples of how these information rich reagents can be used for determining gene function, discovering novel therapeutic targets and dissecting signalling pathways, cellular processes and complex phenotypes.     

Coincidence detection in phosphoinositide signaling

Phosphoinositide lipids function as both signaling molecules and as compartment-specific localization signals for phosphoinositide-binding proteins. In recent years, both phosphoinositides and phosphoinositide-binding proteins have been reported to display a restricted, rather than a uniform, distribution across intracellular membranes. Here, we examine recent data documenting the restricted distribution of both phosphoinositides and phosphoinositide-binding proteins and examine how phosphoinositide-binding proteins might engage multiple binding partners to achieve these restricted localizations, effectively acting as detectors of coincident localization signals.     

Sorting nexins--unifying trends and new perspectives

The sorting nexins (SNXs) are a family of PX domain-containing proteins found in yeast and mammalian cells that have been proposed to regulate intracellular trafficking. Mammalian SNXs have been suggested to function variously in pro-degradative sorting, internalization, endosomal recycling, or simply in endosomal sorting. In yeast, the defining function for these proteins is a regulation of cargo retrieval. Here we examine recent data on the SNX family of proteins and attempt to draw out unifying themes between the work performed in yeast and mammalian systems.