Orally active antischistosomal early leads identified from the open access malaria box.

BACKGROUND: Worldwide hundreds of millions of schistosomiasis patients rely on treatment with a single drug, praziquantel. Therapeutic limitations and the threat of praziquantel resistance underline the need to discover and develop next generation drugs. METHODOLOGY: We studied the antischistosomal properties of the Medicines for Malaria Venture (MMV) malaria box containing 200 diverse drug-like and 200 probe-like compounds with confirmed in vitro activity against Plasmodium falciparum. Compounds were tested against schistosomula and adult Schistosoma mansoni in vitro. Based on in vitro performance, available pharmacokinetic profiles and toxicity data, selected compounds were investigated in vivo. PRINCIPAL FINDINGS: Promising antischistosomal activity (IC50: 1.4-9.5 µM) was observed for 34 compounds against schistosomula. Three compounds presented IC50 values between 0.8 and 1.3 µM against adult S. mansoni. Two promising early leads were identified, namely a N,N'-diarylurea and a 2,3-dianilinoquinoxaline. Treatment of S. mansoni infected mice with a single oral 400 mg/kg dose of these drugs resulted in significant worm burden reductions of 52.5% and 40.8%, respectively. CONCLUSIONS/SIGNIFICANCE: The two candidates identified by investigating the MMV malaria box are characterized by good pharmacokinetic profiles, low cytotoxic potential and easy chemistry and therefore offer an excellent starting point for antischistosomal drug discovery and development

Image informatics strategies for deciphering neuronal network connectivity

Brain function relies on an intricate network of highly dynamic neuronal connections that rewires dramatically under the impulse of various external cues and pathological conditions. Among the neuronal structures that show morphologi- cal plasticity are neurites, synapses, dendritic spines and even nuclei. This structural remodelling is directly connected with functional changes such as intercellular com- munication and the associated calcium-bursting behaviour. In vitro cultured neu- ronal networks are valuable models for studying these morpho-functional changes. Owing to the automation and standardisation of both image acquisition and image analysis, it has become possible to extract statistically relevant readout from such networks. Here, we focus on the current state-of-the-art in image informatics that enables quantitative microscopic interrogation of neuronal networks. We describe the major correlates of neuronal connectivity and present workflows for analysing them. Finally, we provide an outlook on the challenges that remain to be addressed, and discuss how imaging algorithms can be extended beyond in vitro imaging studies

Consequences of simulated microgravity in neural stem cells: biological effects and metabolic response.

Objective: Microgravity was often shown to cause cell damage and impair cell cycle in a variety of biological systems. Since the effects on the neural system were poorly investigated, we aimed to gain insight into how biological processes such as cell cycle, cell damage, stemness features and metabolic status are involved in neural stem cells (NSC) when they experience simulated microgravity. We also wished to investigate whether these modulations were transient or permanent once cells were returned to normal gravity. Methods: NSC were isolated from mouse cerebella and cultured in the Rotary Cell Culture System (RCCS) to model microgravity. We analyzed cell cycle, stress and apoptotic response. We also performed a 1H NMR-based metabolomic analysis and evaluation of stemness features of NSC in simulated microgravity and once in the returned to normogravity cell culture. Results: Biological processes and metabolic status were modulated by simulated microgravity. Cells were arrested in S-phase together with enhanced apoptosis. Metabolic changes occurred in NSC after simulated microgravity. Interestingly, these modulations were transient. Indeed, stemness features and metabolic footprint returned to basal levels after few days of culture in normal conditions. Moreover NSC clonogenic ability was not impaired. Conclusions: Our data suggest that simulated microgravity impacts on NSC biological processes, including cell cycle and apoptosis. However, NSC does not suffer from permanent damage

Assessment of 19 Genes and Validation of CRM Gene Panel for Quantitative Transcriptional Analysis of Molecular Rejection and Inflammation in Archival Kidney Transplant Biopsies.

Background: There is an urgent need to develop and implement low cost, high-throughput standardized methods for routine molecular assessment of transplant biopsies. Given the vast archive of formalin-fixed and paraffin-embedded (FFPE) tissue blocks in transplant centers, a reliable protocol for utilizing this tissue bank for clinical validation of target molecules as predictors of graft outcome over time, would be of great value. Methods: We designed and optimized assays to quantify 19 target genes, including previously reported set of tissue common rejection module (tCRM) genes. We interrogated their performance for their clinical utility for detection of graft rejection and inflammation by analyzing gene expression microarrays analysis of 163 renal allograft biopsies, and subsequently validated in 40 independent FFPE archived kidney transplant biopsies at a single center. Results: A QPCR (Fluidigm) and a barcoded oligo-based (NanoString) gene expression platform were compared for evaluation of amplification of gene expression signal for 19 genes from degraded RNA extracted from FFPE biopsy sections by a set protocol. Increased expression of the selected 19 genes, that reflect a combination of specific cellular infiltrates (8/19 genes) and a graft inflammation score (11/19 genes which computes the tCRM score allowed for segregation of kidney transplant biopsies with stable allograft function and normal histology from those with histologically confirmed acute rejection (AR; p = 0.0022, QPCR; p = 0.0036, barcoded assay) and many cases of histological borderline inflammation (BL). Serial biopsy shaves used for gene expression were also processed for in-situ hybridization (ISH) for a subset of genes. ISH confirmed a high degree of correlation of signal amplification and tissue localization. Conclusions: Target gene expression amplification across a custom set of genes can identify AR independent of histology, and quantify inflammation from archival kidney transplant biopsy tissue, providing a new tool for clinical correlation and outcome analysis of kidney allografts, without the need for prospective kidney biopsy biobanking efforts

Caveolin-1 protects B6129 mice against Helicobacter pylori gastritis.

Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies ("humming bird") compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells

Cyclin B1/Cdk1 phosphorylation of mitochondrial p53 induces anti-apoptotic response.

The pro-apoptotic function of p53 has been well defined in preventing genomic instability and cell transformation. However, the intriguing fact that p53 contributes to a pro-survival advantage of tumor cells under DNA damage conditions raises a critical question in radiation therapy for the 50% human cancers with intact p53 function. Herein, we reveal an anti-apoptotic role of mitochondrial p53 regulated by the cell cycle complex cyclin B1/Cdk1 in irradiated human colon cancer HCT116 cells with p53(+/+) status. Steady-state levels of p53 and cyclin B1/Cdk1 were identified in the mitochondria of many human and mouse cells, and their mitochondrial influx was significantly enhanced by radiation. The mitochondrial kinase activity of cyclin B1/Cdk1 was found to specifically phosphorylate p53 at Ser-315 residue, leading to enhanced mitochondrial ATP production and reduced mitochondrial apoptosis. The improved mitochondrial function can be blocked by transfection of mutant p53 Ser-315-Ala, or by siRNA knockdown of cyclin B1 and Cdk1 genes. Enforced translocation of cyclin B1 and Cdk1 into mitochondria with a mitochondrial-targeting-peptide increased levels of Ser-315 phosphorylation on mitochondrial p53, improved ATP production and decreased apoptosis by sequestering p53 from binding to Bcl-2 and Bcl-xL. Furthermore, reconstitution of wild-type p53 in p53-deficient HCT116 p53(-/-) cells resulted in an increased mitochondrial ATP production and suppression of apoptosis. Such phenomena were absent in the p53-deficient HCT116 p53(-/-) cells reconstituted with the mutant p53. These results demonstrate a unique anti-apoptotic function of mitochondrial p53 regulated by cyclin B1/Cdk1-mediated Ser-315 phosphorylation in p53-wild-type tumor cells, which may provide insights for improving the efficacy of anti-cancer therapy, especially for tumors that retain p53