Discovery of a novel aminopyrazine series as selective PI3Kα inhibitors

We report the discovery of a novel aminopyrazine series of PI3Kα inhibitors, designed by hybridizing two known scaffolds of PI3K inhibitors. We describe the progress achieved from the first compounds plagued with poor general kinase selectivity to compounds showing high selectivity for PI3Kα over PI3Kβ and excellent general kinase selectivity. This effort culminated with the identification of compound 5 displaying high potency and selectivity, and suitable physiochemical and pharmacokinetic properties for oral administration. In vivo, compound 5 showed good inhibition of tumour growth (86% tumour growth inhibition at 50 mg/kg twice daily orally) in the MCF7 xenograft model in mice.     

SELDI-TOF MS detection of urinary hepcidin

Hepcidin is a 25-residue hepatic peptide that regulates iron absorption from the diet and tissue iron distribution. Inappropriately low Hepcidin expression is implicated in the pathogenesis of hereditary hemochromatosis and iron-loading anemias, like the thalassemias. Increased hepcidin expression mediates iron retention in the anemias of inflammation and plays a pathogenic role in iron-refractory iron-deficiency anemia (IRIDA). Because of its clinical importance, Hepcidin is expected to be a useful biomarker for diagnosis and management of iron-related disorders. So far an ELISA for human hepcidin and SELDI-TOF-MS based approaches have been applied to monitor urinary and/or serum hepcidin levels. Here we report a modified protocol for SELDI-TOF based detection of human, urinary hepcidin. We show that CM10 Proteinchips are superior to NP20 Proteinchips commonly used in previously reported protocols to sensitively and accurately detect urinary hepcidin. Application of this modified hepcidin assay accurately detects increased hepcidin levels in the urine of sepsis patients.     

Melanoma Cells Treated with GGTI and IFN-γ Allow Murine Vaccination and Enhance Cytotoxic Response against Human Melanoma Cells

Background

Suboptimal activation of T lymphocytes by melanoma cells is often due to the defective expression of class I major histocompatibility antigens (MHC-I) and costimulatory molecules. We have previously shown that geranylgeranyl transferase inhibition (done with GGTI-298) stimulates anti-melanoma immune response through MHC-I and costimulatory molecule expression in the B16F10 murine model [1].

Methodology/Principal Findings

In this study, it is shown that vaccination with mIFN-gand GGTI-298 pretreated B16F10 cells induces a protection against untreated tumor growth and pulmonary metastases implantation. Furthermore, using a human melanoma model (LB1319-MEL), we demonstrated that in vitro treatment with hIFN-γ and GGTI-298 led to the up regulation of MHC-I and a costimulatory molecule CD86 and down regulation of an inhibitory molecule PD-1L. Co-culture experiments with peripheral blood mononuclear cells (PBMC) revealed that modifications induced by hIFN-γ and GGTI-298 on the selected melanoma cells, enables the stimulation of lymphocytes from HLA compatible healthy donors. Indeed, as compared with untreated melanoma cells, pretreatment with hIFN-γ and GGTI-298 together rendered the melanoma cells more efficient at inducing the: i) activation of CD8 T lymphocytes (CD8+/CD69+); ii) proliferation of tumor-specific CD8 T cells (MelanA-MART1/TCR+); iii) secretion of hIFN-γ; and iv) anti-melanoma specific cytotoxic cells.

Conclusions/Significance

These data indicate that pharmacological treatment of melanoma cell lines with IFN-γ and GGTI-298 stimulates their immunogenicity and could be a novel approach to produce tumor cells suitable for vaccination and for stimulation of anti-melanoma effector cells.     

A tandem repeats database for bacterial genomes: application to the genotyping of Yersinia pestis and Bacillus anthracis

Background  

Some pathogenic bacteria are genetically very homogeneous, making strain discrimination difficult. In the last few years, tandem repeats have been increasingly recognized as markers of choice for genotyping a number of pathogens. The rapid evolution of these structures appears to contribute to the phenotypic flexibility of pathogens. The availability of whole-genome sequences has opened the way to the systematic evaluation of tandem repeats diversity and application to epidemiological studies.     

Detection by DGGE of a new polymorphism closely linked to the adenomatous polyposis coli region

Summary The EF5.44 locus is in close proximity to the chromosome 5 region to which the genetic defect responsible for familial adenomatous polyposis has been mapped. We have devised two oligonucleotides that promote the specific polymerase chain reaction (PCR) amplificiation of a 365-bp sequence in this region. Analysis by denaturing gradient gel electrophoresis of the resulting fragment has unravelled individual differences that could be identified as a single base pair change in aMnlI restriction site. This PCR assayable polymorphism increases the informativeness at this locus, and should be useful in the presymptomatic diagnosis of familial adenomatous polyposis.     

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.     

Quantitative trait loci (QTL) detection in multicross inbred designs: recovering QTL identical-by-descent status information from marker data

Mapping quantitative trait loci in plants is usually conducted using a population derived from a cross between two inbred lines. The power of such QTL detection and the parameter estimates depend largely on the choice of the two parental lines. Thus, the QTL detected in such populations represent only a small part of the genetic architecture of the trait. In addition, the effects of only two alleles are characterized, which is of limited interest to the breeder, while common pedigree breeding material remains unexploited for QTL mapping. In this study, we extend QTL mapping methodology to a generalized framework, based on a two-step IBD variance component approach, applicable to any type of breeding population obtained from inbred parents. We then investigate with simulated data mimicking conventional breeding programs the influence of different estimates of the IBD values on the power of QTL detection. The proposed method would provide an alternative to the development of specifically designed recombinant populations, by utilizing the genetic variation actually managed by plant breeders. The use of these detected QTL in assisting breeding would thus be facilitated.     

Development of a lab-made microarray for analyzing the genetic diversity of nitrogen fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.     

Monotonicity of interleukin-1 receptor-ligand binding with respect to antagonist in the presence of decoy receptor

We consider the interaction between interleukin-1 IL-1, its receptor IL-1RI, the receptor antagonist IL-1Ra and a decoy receptor (or trap) that binds both with the ligand and the antagonist. We study how the interaction between IL-1Ra and the decoy receptor influences the effect of either reagent on reducing the equilibrium concentration of the receptor-ligand complex. We obtain that, given a certain relationship among the equilibrium constants and the total concentrations of solutes, IL-1Ra can reverse the effect of the decoy receptor of decreasing the equilibrium concentration of the receptor-ligand complex. This finding derives from a mathematical result applicable to any reversible chemical reaction system comprising four species arranged in a square such that each species binds its two immediate neighbors. The result gives the monotonicity of the equilibrium concentrations of the complex species as functions of the total concentrations of the simple species.     

Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women

Cervicovaginal microbiota not dominated by lactobacilli may facilitate transmission of HIV and other sexually transmitted infections (STIs), as well as miscarriages, preterm births and sepsis in pregnant women. However, little is known about the exact nature of the microbiological changes that cause these adverse outcomes. In this study, cervical samples of 174 Rwandan female sex workers were analyzed cross-sectionally using a phylogenetic microarray. Furthermore, HIV-1 RNA concentrations were measured in cervicovaginal lavages of 58 HIV-positive women among them. We identified six microbiome clusters, representing a gradient from low semi-quantitative abundance and diversity dominated by Lactobacillus crispatus (cluster R-I, with R denoting ‘Rwanda'') and L. iners (R-II) to intermediate (R-V) and high abundance and diversity (R-III, R-IV and R-VI) dominated by a mixture of anaerobes, including Gardnerella, Atopobium and Prevotella species. Women in cluster R-I were less likely to have HIV (P=0.03), herpes simplex virus type 2 (HSV-2; P<0.01), and high-risk human papillomavirus (HPV; P<0.01) and had no bacterial STIs (P=0.15). Statistically significant trends in prevalence of viral STIs were found from low prevalence in cluster R-I, to higher prevalence in clusters R-II and R-V, and highest prevalence in clusters R-III/R-IV/R-VI. Furthermore, only 10% of HIV-positive women in clusters R-I/R-II, compared with 40% in cluster R-V, and 42% in clusters R-III/R-IV/R-VI had detectable cervicovaginal HIV-1 RNA (P_trend=0.03). We conclude that L. crispatus-dominated, and to a lesser extent L. iners-dominated, cervicovaginal microbiota are associated with a lower prevalence of HIV/STIs and a lower likelihood of genital HIV-1 RNA shedding.