A theoretical entropy score as a single value to express inhibitor selectivity

Background  

Designing maximally selective ligands that act on individual targets is the dominant paradigm in drug discovery. Poor selectivity can underlie toxicity and side effects in the clinic, and for this reason compound selectivity is increasingly monitored from very early on in the drug discovery process. To make sense of large amounts of profiling data, and to determine when a compound is sufficiently selective, there is a need for a proper quantitative measure of selectivity.     

MicroRNA‐155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft‐cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA‐155 (miR‐155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4‐fold increase of miR‐155 when CMPCs were exposed to hydrogen‐peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR‐155 in CMPCs revealed that miR‐155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre‐incubating the cells with a RIP1 specific inhibitor, Necrostatin‐1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR‐array showed that increased miR‐155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR‐155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro‐survival pathway. MiR‐155 provides the opportunity to block necrosis, a conventionally thought non‐regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.     

GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

Background

Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.

Methodology/Principal Findings

We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1^−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.

Conclusions/Significance

Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.     

The Slc11a1 (Nramp1) gene controls efficacy of mycobacterial treatment of allergic asthma

Genes controlling antibacterial resistance may be important in the hygiene hypothesis, which states that lack of bacterial infections during childhood would favor development of allergic disease. We, therefore, studied whether Nramp1 (Slc11a1) alleles, which determine susceptibility (Nramp1(s)) or resistance (Nramp1(r)) to intracellular bacteria, affect the efficacy of heat-killed Mycobacterium vaccae in the treatment of allergic asthma in a mouse model. Treatment of OVA-sensitized Nramp1(s) mice with M. vaccae suppressed airway hyperresponsiveness, airway eosinophilia, Ag-specific IgE, and IL-4 and IL-5 production after OVA aerosol challenge. In contrast, M. vaccae hardly affected these parameters in Nramp1(r) mice. In addition, The Nramp1 gene affected both T cell-mediated responses to M. vaccae in vivo and the level of macrophage activation after stimulation with M. vaccae in vitro. In conclusion, the efficacy of M. vaccae in preventing allergic and asthmatic manifestations in a mouse model is strongly affected by Nramp1 alleles. These findings could have important implications for the future use of mycobacteria and their components in the prevention or treatment of allergic asthma. A new link is described between genes, the environment, and the development of allergy, in which the Nramp1 gene fine tunes the hygiene hypothesis.     

Novel peptides based on HIV-1 gp120 sequence with homology to chemokines inhibit HIV infection in cell culture

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.     

Differential transmission of human immunodeficiency virus type 1 by distinct subsets of effector dendritic cells

Dendritic cells (DC) support human immunodeficiency virus type 1 (HIV-1) transmission by capture of the virus particle in the mucosa and subsequent transport to the draining lymph node, where HIV-1 is presented to CD4(+) Th cells. Virus transmission involves a high-affinity interaction between the DC-specific surface molecule DC-SIGN and the viral envelope glycoprotein gp120 and subsequent internalization of the virus, which remains infectious. The mechanism of viral transmission from DC to T cells is currently unknown. Sentinel immature DC (iDC) develop into Th1-promoting effector DC1 or Th2-promoting DC2, depending on the activation signals. We studied the ability of these effector DC subsets to support HIV-1 transmission in vitro. Compared with iDC, virus transmission is greatly upregulated for the DC1 subset, whereas DC2 cells are inactive. Increased transmission by DC1 correlates with increased expression of ICAM-1, and blocking studies confirm that ICAM-1 expression on DC is important for HIV transmission. The ICAM-1-LFA-1 interaction is known to be important for immunological cross talk between DC and T cells, and our results indicate that this cell-cell contact is exploited by HIV-1 for efficient transmission.     

Monocyte Chemoattractant Protein-1/CCL2 Produced by Stromal Cells Promotes Lung Metastasis of 4T1 Murine Breast Cancer Cells

MCP-1/CCL2 plays an important role in the initiation and progression of cancer. Since tumor cells produce MCP-1, they are considered to be the main source of this chemokine. Here, we examined whether MCP-1 produced by non-tumor cells affects the growth and lung metastasis of 4T1 breast cancer cells by transplanting them into the mammary pad of WT or MCP-1^−/− mice. Primary tumors at the injected site grew similarly in both mice; however, lung metastases were markedly reduced in MCP-1^−/− mice, with significantly longer mouse survival. High levels of MCP-1 mRNA were detected in tumors growing in WT, but not MCP-1^−/− mice. Serum MCP-1 levels were increased in tumor-bearing WT, but not MCP-1^−/− mice. Transplantation of MCP-1^−/− bone marrow cells into WT mice did not alter the incidence of lung metastasis, whereas transplantation of WT bone marrow cells into MCP-1^−/− mice increased lung metastasis. The primary tumors of MCP-1^−/− mice consistently developed necrosis earlier than those of WT mice and showed decreased infiltration by macrophages and reduced angiogenesis. Interestingly, 4T1 cells that metastasized to the lung constitutively expressed elevated levels of MCP-1, and intravenous injection of 4T1 cells producing a high level of MCP-1 resulted in increased tumor foci in the lung of WT and MCP-1^−/− mice. Thus, stromal cell-derived MCP-1 in the primary tumors promotes lung metastasis of 4T1 cells, but tumor cell-derived MCP-1 can also contribute once tumor cells enter the circulation. A greater understanding of the source and role of this chemokine may lead to novel strategies for cancer treatment.     

Gliotactin,a novel marker of tricellular junctions,is necessary for septate junction development in Drosophila

Septate junctions (SJs), similar to tight junctions, function as transepithelial permeability barriers. Gliotactin (Gli) is a cholinesterase-like molecule that is necessary for blood-nerve barrier integrity, and may, therefore, contribute to SJ development or function. To address this hypothesis, we analyzed Gli expression and the Gli mutant phenotype in Drosophila epithelia. In Gli mutants, localization of SJ markers neurexin-IV, discs large, and coracle are disrupted. Furthermore, SJ barrier function is lost as determined by dye permeability assays. These data suggest that Gli is necessary for SJ formation. Surprisingly, Gli distribution only colocalizes with other SJ markers at tricellular junctions, suggesting that Gli has a unique function in SJ development. Ultrastructural analysis of Gli mutants supports this notion. In contrast to other SJ mutants in which septa are missing, septa are present in Gli mutants, but the junction has an immature morphology. We propose a model, whereby Gli acts at tricellular junctions to bind, anchor, or compact SJ strands apically during SJ development.     

NRMD: Nuclear Receptor Mutation Database

The NRMD is a database for nuclear receptor mutation information. It includes mutation information from SWISS-PROT/TrEMBL, several web-based mutation data resources, and data extracted from the literature in a fully automatic manner. Because it is also possible to add mutations manually, a hundred mutations were added for completeness. At present, the NRMD contains information about 893 mutations in 54 nuclear receptors. A common numbering scheme for all nuclear receptors eases the use of the information for many kinds of studies. The NRMD is freely available to academia and industry as a stand-alone version at: www.receptors.org/NR/.     

Differential modes of MHC class IIB gene evolution in cichlid fishes

Cichlid fishes are emblematic models for the study of adaptive radiation, driven by natural and sexual selection. Parasite mediated selection is an important component in these processes, and the evolution of their immune system therefore merits special attention. In this study, light is shed on the phylogeny of the b family of cichlid major histocompatibility complex (MHC) class IIB genes. Full-length coding sequences were used to reconstruct phylogenies using criteria of maximum parsimony, maximum likelihood and Bayesian inference. All analyses suggest monophyly of the b family of cichlid MHC class IIB genes, although sequences of the cichlid sister taxa are currently not available. Two evolutionary lineages of these genes, respectively encompassing the recently defined genomic regions DBB-DEB-DFB and DCB-DDB, show highly contrasting levels of differentiation. To explore putative causes for these differences, exon 2 sequences were screened for variation in recombination rate and strength of selection. The more diversified lineage of cichlid MHC class IIB b genes was found to have higher levels of both recombination and selection. This is consistent with the observation in other taxa that recombination facilitates the horizontal spread of positively selected sites across MHC loci and hence contributes to fast sequence evolution. In contrast, the lineage that showed low diversification might either be under stabilizing selection or is evolutionary constrained by its low recombination rate. We speculate whether this lineage might include MHC genes with non-classical functions.