Multivalent helix mimetics for PPI-inhibition.

The exploitation of multivalent ligands for the inhibition of protein-protein interactions has not yet been explored as a supramolecular design strategy. This is despite the fact that protein-protein interactions typically occur within the context of multi-protein complexes and frequently exploit avidity effects or co-operative binding interactions to achieve high affinity interactions. In this paper we describe preliminary studies on the use of a multivalent N-alkylated aromatic oligoamide helix mimetic for inhibition of p53/hDM2 and establish that protein dimerisation is promoted, rather than enhanced binding resulting from a higher effective concentration of the ligand. This journal i

Regulatory targets of quorum sensing in Vibrio cholerae: evidence for two distinct HapR-binding motifs

The quorum-sensing pathway in Vibrio cholerae controls the expression of the master regulator HapR, which in turn regulates several important processes such as virulence factor production and biofilm formation. While HapR is known to control several important phenotypes, there are only a few target genes known to be transcriptionally regulated by HapR. In this work, we combine bioinformatic analysis with experimental validation to discover a set of novel direct targets of HapR. Our results provide evidence for two distinct binding motifs for HapR-regulated genes in V. cholerae. The first binding motif is similar to the motifs recently discovered for orthologs of HapR in V. harveyi and V. vulnificus. However, our results demonstrate that this binding motif can be of variable length in V. cholerae. The second binding motif shares common elements with the first motif, but is of fixed length and lacks dyad symmetry at the ends. The contributions of different bases to HapR binding for this second motif were demonstrated using systematic mutagenesis experiments. The current analysis presents an approach for systematically expanding our knowledge of the quorum-sensing regulon in V. cholerae and other related bacteria

Improving the utility of evidence synthesis for decision makers in the face of insufficient evidence.

OBJECTIVE: To identify and suggest strategies to make insufficient evidence ratings in systematic reviews more actionable. STUDY DESIGN AND SETTING: A workgroup comprising members from the Evidence-Based Practice (EPC) Program of the Agency for Healthcare Research and Quality convened throughout 2020. We conducted iterative discussions considering information from three data sources: a literature review for relevant publications and frameworks, a review of a convenience sample of past systematic reviews conducted by the EPCs, and an audit of methods used in past EPC technical briefs. RESULTS: We identified five strategies for supplementing systematic review findings when evidence on benefits or harms is expected to be, or found to be, insufficient: 1) reconsider eligible study designs, 2) summarize indirect evidence, 3) summarize contextual and implementation evidence, 4) consider modelling, and 5) incorporate unpublished health system data in the evidence synthesis. While these strategies may not increase the strength of evidence, they may improve the utility of reports for decision makers. Adopting these strategies depends on feasibility, timeline, funding, and expertise of the systematic reviewers. CONCLUSION: Throughout the process of evidence synthesis of early scoping, protocol development, review conduct, and review presentation, authors can consider these five strategies to supplement evidence with insufficient rating to make it more actionable for end-users

Control of Centrin Stability by Aurora A

Aurora A is an oncogenic serine/threonine kinase which can cause cell transformation and centrosome amplification when over-expressed. Human breast tumors show excess Aurora A and phospho-centrin in amplified centrosomes. Here, we show that Aurora A mediates the phosphorylation of and localizes with centrin at the centrosome, with both proteins reaching maximum abundance from prophase through metaphase, followed by their precipitous loss in late stages of mitosis. Over-expression of Aurora A results in excess phospho-centrin and centrosome amplification. In contrast, centrosome amplification is not seen in cells over-expressing Aurora A in the presence of a recombinant centrin mutant lacking the serine phosphorylation site at residue 170. Expression of a kinase dead Aurora A results in a decrease in mitotic index and abrogation of centrin phosphorylation. Finally, a recombinant centrin mutation that mimics centrin phosphorylation increases centrin's stability against APC/C-mediated proteasomal degradation. Taken together, these results suggest that the stability of centrin is regulated in part by Aurora A, and that excess phosphorylated centrin may promote centrosome amplification in cancer

Novel targets and a cofactor for the Vibrio cholerae quorum sensing transcriptional regulator, HapR

Vibrio cholerae, which causes the severely dehydrating diarrheal disease, cholera, undergoes drastic changes in living conditions as it moves between its aquatic reservoir and its human host. These different living conditions require different gene expression profiles. One method that V. cholerae uses to sense changes in the environment, particularly changes in cell density, is quorum sensing. The bacteria produce small molecules called autoinducers that accumulate with increasing cell density and trigger intracellular signaling pathways to alter gene expression. These intracellular signaling pathways regulate the expression level of the transcriptional regulator, HapR, which controls many phenotypes including virulence factor production and biofilm formation. The studies presented here provide insight into the phenotypes that HapR regulates and the mechanism by which HapR provides this regulation. A bioinformatics-based approach was used to determine consensus binding sequences for HapR and to identify novel targets that are directly regulated by HapR. Many new HapR-regulated promoters were found, and some were activated while others were repressed by HapR. This study led us to investigate HapR\u27s regulation of a hemolysin, HlyA, that had previously been shown to be a potential virulence factor. HapR was found to repress HlyA on two levels: one by directly binding to and repressing transcription from its promoter and another by inducing the transcription of a protease, HapA, which degrades the HlyA protein. HapR is known to repress V. cholerae\u27s two main virulence factors, cholera toxin and the toxin-coregulated pilus, and so its regulation of HlyA represents another mechanism by which quorum sensing represses virulence. The third study identifies a two-component system, VarS/VarA, that is required for full HapR function. A previous report showed that VarS and VarA regulate HapR expression through the quorum sensing system, but we show that in addition to this, VarS and VarA also modulate HapR activity. Since VarS and VarA affect HapR\u27s regulation of its targets in two ways, it is likely that the integration of VarS/VarA signaling with quorum sensing is important. We propose that this enables V. cholerae to combine cell density and nutrient availability information to more appropriately control gene expression

Novel targets and a cofactor for the Vibrio cholerae quorum sensing transcriptional regulator, HapR

Vibrio cholerae, which causes the severely dehydrating diarrheal disease, cholera, undergoes drastic changes in living conditions as it moves between its aquatic reservoir and its human host. These different living conditions require different gene expression profiles. One method that V. cholerae uses to sense changes in the environment, particularly changes in cell density, is quorum sensing. The bacteria produce small molecules called autoinducers that accumulate with increasing cell density and trigger intracellular signaling pathways to alter gene expression. These intracellular signaling pathways regulate the expression level of the transcriptional regulator, HapR, which controls many phenotypes including virulence factor production and biofilm formation. The studies presented here provide insight into the phenotypes that HapR regulates and the mechanism by which HapR provides this regulation. A bioinformatics-based approach was used to determine consensus binding sequences for HapR and to identify novel targets that are directly regulated by HapR. Many new HapR-regulated promoters were found, and some were activated while others were repressed by HapR. This study led us to investigate HapR\u27s regulation of a hemolysin, HlyA, that had previously been shown to be a potential virulence factor. HapR was found to repress HlyA on two levels: one by directly binding to and repressing transcription from its promoter and another by inducing the transcription of a protease, HapA, which degrades the HlyA protein. HapR is known to repress V. cholerae\u27s two main virulence factors, cholera toxin and the toxin-coregulated pilus, and so its regulation of HlyA represents another mechanism by which quorum sensing represses virulence. The third study identifies a two-component system, VarS/VarA, that is required for full HapR function. A previous report showed that VarS and VarA regulate HapR expression through the quorum sensing system, but we show that in addition to this, VarS and VarA also modulate HapR activity. Since VarS and VarA affect HapR\u27s regulation of its targets in two ways, it is likely that the integration of VarS/VarA signaling with quorum sensing is important. We propose that this enables V. cholerae to combine cell density and nutrient availability information to more appropriately control gene expression

Quorum Sensing Negatively Regulates Hemolysin Transcriptionally and Posttranslationally in Vibrio cholerae▿

Recent work has shown that in addition to cholera toxin (CT) and the toxin-coregulated pilus (TCP), other cytotoxic proteins in Vibrio cholerae also cause disease symptoms, and this is particularly evident in strains lacking CT. One such protein is the hemolysin encoded by hlyA. Here we show that, like CT and TCP, HlyA is repressed by the quorum-sensing-regulated transcription factor HapR. This repression occurs on two levels: one at the transcriptional level that is independent of the metalloprotease HapA and one at the posttranslational level that is mediated by HapA. The transcriptional regulation is significantly more apparent on solid media than in liquid cultures. This is the first time that hemolysis has been shown to be directly regulated by quorum sensing in V. cholerae, and it is interesting that, like other virulence factors, HlyA is also repressed by HapR, which is expressed late in infection