A novel FRET-based screen in high-throughput format to identify inhibitors of malarial and human glucose transporters

The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z′ factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum. Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC(50)) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening

Preserved Edge Convolutional Neural Network for Sensitivity Enhancement of Deuterium Metabolic Imaging (DMI)

Purpose: Common to most MRSI techniques, the spatial resolution and the minimal scan duration of Deuterium Metabolic Imaging (DMI) are limited by the achievable SNR. This work presents a deep learning method for sensitivity enhancement of DMI. Methods: A convolutional neural network (CNN) was designed to estimate the 2H-labeled metabolite concentrations from low SNR and distorted DMI FIDs. The CNN was trained with synthetic data that represent a range of SNR levels typically encountered in vivo. The estimation precision was further improved by fine-tuning the CNN with MRI-based edge-preserving regularization for each DMI dataset. The proposed processing method, PReserved Edge ConvolutIonal neural network for Sensitivity Enhanced DMI (PRECISE-DMI), was applied to simulation studies and in vivo experiments to evaluate the anticipated improvements in SNR and investigate the potential for inaccuracies. Results: PRECISE-DMI visually improved the metabolic maps of low SNR datasets, and quantitatively provided higher precision than the standard Fourier reconstruction. Processing of DMI data acquired in rat brain tumor models resulted in more precise determination of 2H-labeled lactate and glutamate + glutamine levels, at increased spatial resolution (from >8 to 2 $\mu$L) or shortened scan time (from 32 to 4 min) compared to standard acquisitions. However, rigorous SD-bias analyses showed that overuse of the edge-preserving regularization can compromise the accuracy of the results. Conclusion: PRECISE-DMI allows a flexible trade-off between enhancing the sensitivity of DMI and minimizing the inaccuracies. With typical settings, the DMI sensitivity can be improved by 3-fold while retaining the capability to detect local signal variations

Tropospheric aqueous-phase chemistry: kinetics, mechanisms, and its coupling to a changing gas phase

[no abstract available

Varied effects of algal symbionts on transcription factor NF-κB in a sea anemone and a coral: possible roles in symbiosis and thermotolerance

Many cnidarians, including the reef-building corals, undergo symbiotic mutualisms with photosynthetic dinoflagellate algae of the family Symbiodiniaceae. These partnerships are sensitive to temperature extremes, which cause symbiont loss and increased coral mortality. Previous studies have implicated host immunity and specifically immunity transcription factor NF-κB as having a role in the maintenance of the cnidarian-algal symbiosis. Here we have further investigated a possible role for NF-κB in establishment and loss of symbiosis in various strains of the anemone Exaiptasia (Aiptasia) and in the coral Pocillopora damicornis. Our results show that NF-κB expression is reduced in Aiptasia larvae and adults that host certain algae strains. Treatment of Aiptasia larvae with a known symbiosis-promoting cytokine, transforming growth factor β, also led to decreased NF-κB expression. We also show that aposymbiotic Aiptasia (with high NF-κB expression) have increased survival following infection with the pathogenic bacterium Serratia marcescens as compared to symbiotic Aiptasia (low NF-κB expression). Furthermore, a P. damicornis coral colony hosting Durusdinium spp. (formerly clade D) symbionts had higher basal NF-κB expression and decreased heat-induced bleaching as compared to two individuals hosting Cladocopium spp. (formerly clade C) symbionts. Lastly, genome-wide gene expression profiling and genomic promoter analysis identified putative NF-κB target genes that may be involved in thermal bleaching, symbiont maintenance, and/or immune protection in P. damicornis. Our results provide further support for the hypothesis that modulation of NF-κB and immunity plays a role in some, but perhaps not all, cnidarian-Symbiodiniaceae partnerships as well as in resistance to pathogens and bleaching.Accepted manuscrip

Introducción

There are miraculous encounters. Under the good auspices of Cartagena de Indias, collaboration began between the Francisco José de Caldas District University (Colombia) and the Jean-Jaurès University of Toulouse (France). In the framework of a meeting organized by the Colombian Association of Universities (ASCUN) entitled "A contribution to public policy on Doctoral training in Colombia", the University of Toulouse was invited to present the organization of the doctoral diploma in March 2015 in France, following a project newly coined in Toulouse as the School of Doctors. This school that brings together 4,500 doctoral students and 7,000 researchers could be an example to enhance the dynamics of research and innovation through doctoral studentsHay encuentros milagrosos. Bajo los buenos auspicios de Cartagena de Indias empezó la colaboración entre la Universidad Distrital Francisco José de Caldas (Colombia) y la Universidad de Toulouse Jean-Jaurès (Francia). En el marco de un encuentro organizado por la Asociación Colombiana de Universidades (ASCUN) titulada “Un aporte a la política pública sobre la formación Doctoral en Colombia”, la Universidad de Toulouse fue invitada a presentar en marzo del 2015 la organización del diploma de doctorado en Francia, a raíz de un proyecto nuevamente acuñado en Toulouse como Escuela de Doctores. Esta escuela que reúne a 4.500 doctorandos y 7.000 investigadores podía ser un ejemplo para potenciar la dinámica de la investigación e innovación a través de los doctorandosExistem encontros milagrosos. Sob os bons auspícios de Cartagena das Índias, a colaboração começou entre a Universidade do Distrito Francisco José de Caldas (Colômbia) e a Universidade Jean-Jaurès de Toulouse (França). No âmbito de uma reunião organizada pela Associação Colombiana de Universidades (ASCUN) intitulada "Uma contribuição para as políticas públicas de formação de doutorado na Colômbia", a Universidade de Toulouse foi convidada a apresentar a organização do diploma de doutorado em março de 2015 na França, na sequência de um projeto recentemente cunhado em Toulouse como a Escola de Médicos. Esta escola que reúne 4.500 doutorandos e 7.000 pesquisadores pode ser um exemplo para aprimorar a dinâmica da pesquisa e inovação por meio de doutorando

A Functional Role for ADAM10 in Human Immunodeficiency Virus Type-1 Replication

<p>Abstract</p> <p>Background</p> <p>Gene trap insertional mutagenesis was used as a high-throughput approach to discover cellular genes participating in viral infection by screening libraries of cells selected for survival from lytic infection with a variety of viruses. Cells harboring a disrupted <it>ADAM10 </it>(A Disintegrin and Metalloprotease 10) allele survived reovirus infection, and subsequently ADAM10 was shown by RNA interference to be important for replication of HIV-1.</p> <p>Results</p> <p>Silencing ADAM10 expression with small interfering RNA (siRNA) 48 hours before infection significantly inhibited HIV-1 replication in primary human monocyte-derived macrophages and in CD4⁺cell lines. In agreement, ADAM10 over-expression significantly increased HIV-1 replication. ADAM10 down-regulation did not inhibit viral reverse transcription, indicating that viral entry and uncoating are also independent of ADAM10 expression. Integration of HIV-1 cDNA was reduced in ADAM10 down-regulated cells; however, concomitant 2-LTR circle formation was not detected, suggesting that HIV-1 does not enter the nucleus. Further, ADAM10 silencing inhibited downstream reporter gene expression and viral protein translation. Interestingly, we found that while the metalloprotease domain of ADAM10 is not required for HIV-1 replication, ADAM15 and γ-secretase (which proteolytically release the extracellular and intracellular domains of ADAM10 from the plasma membrane, respectively) do support productive infection.</p> <p>Conclusions</p> <p>We propose that ADAM10 facilitates replication at the level of nuclear trafficking. Collectively, our data support a model whereby ADAM10 is cleaved by ADAM15 and γ-secretase and that the ADAM10 intracellular domain directly facilitates HIV-1 nuclear trafficking. Thus, ADAM10 represents a novel cellular target class for development of antiretroviral drugs.</p

Thrombopoietin: A Novel Bone Healing Agent

poster abstractCritical-size defects in bones do not heal spontaneously and usually require the use of grafts. Unfortunately, grafts have several limitations. To improve bone formation, many clinicians now use bone morphogenetic proteins (BMP), particularly in spinal fusion, fracture healing, and in critical-size defect regeneration. However, multiple side effects of BMP treatment have been uncovered including increased incidence of cancer. Thus, there is great interest in alternatives that allow for safe and effective bone regeneration. Here we show the ability of thrombopoietin (TPO), the main megakaryocyte growth factor, to heal critical-size femoral defects rodents. 5mm or 4mm segmental defects were created in the femur of Long Evans rats or C57BL/6 mice, respectively. The defects were filled with a novel bioabsorbable scaffold which was loaded with recombinant human TPO, BMP-2, or saline, and held stable by a retrograde 1.6 mm intramedullary Kirschner wire (rats) or 23G needle (mice). Xrays were taken every 3 weeks in rats and weekly in mice. Animal were sacrificed at 15 weeks, at which time micro-computed tomography (μCT) and histological analyses were performed. The results observed in mice and rats were similar. The saline control group did not show bridging callus at any time. Both the BMP-2 and TPO groups healed the defect, although bridging callus was evident at earlier times in the BMP-2 groups. However, the TPO groups showed a much more remodeled and physiologic contour on both Xray and μCT. μCT and histological analysis confirms that compared to BMP-2, TPO-treated specimens have a thicker cortex but smaller diameter and smoother contour. TPO appears to restore the original bone contour by stimulating osteoblastogenesis, allowing for periosteal bridging and stabilization to occur, while simultaneously stimulating osteoclast formation. Thus, TPO may serve as a novel bone healing agent

A Study of the PDGF Signaling Pathway with PRISM

In this paper, we apply the probabilistic model checker PRISM to the analysis of a biological system -- the Platelet-Derived Growth Factor (PDGF) signaling pathway, demonstrating in detail how this pathway can be analyzed in PRISM. We show that quantitative verification can yield a better understanding of the PDGF signaling pathway.Comment: In Proceedings CompMod 2011, arXiv:1109.104