Search

Translation of pharmacological results from in vitro cell testing to clinical trials is challenging. One of the causes that may underlie these discrepant results is the lack of the phenotypic or species-specific relevance of the tested cells; today, this lack of relevance may be reduced by relying on cells differentiated from human pluripotent stem cells. To analyse the benefits provided by this approach, we chose to focus on Friedreich ataxia, a neurodegenerative condition for which the recent clinical testing of two compounds was not successful. These compounds, namely, resveratrol and nicotinamide, were selected because they had been shown to stimulate the expression of frataxin in fibroblasts and lymphoblastoid cells. Our results indicated that these compounds failed to do so in iPSC-derived neurons generated from two patients with Friedreich ataxia. By comparing the effects of both molecules on different cell types that may be considered to be non-relevant for the disease, such as fibroblasts, or more relevant to the disease, such as neurons differentiated from iPSCs, a differential response was observed; this response suggests the importance of developing more predictive in vitro systems for drug discovery. Our results demonstrate the value of utilizing human iPSCs early in drug discovery to improve translational predictability.

Communication between gut microbiota and the brain is an enigma. Alterations in the gut microbial community affects enteric metabolite levels, such as short chain fatty acids (SCFAs). SCFAs have been proposed as a possible mechanism through which the gut microbiome modulate brain health and function. This study analyzed for the first time the effects of SCFAs at levels reported in human systemic circulation on SH-SY5Y human neuronal cell energy metabolism, viability, survival, and the brain lipidome. Cell and rat brain lipidomics was done using high resolution mass spectrometry (HRMS). Neuronal cells viability, survival and energy metabolism were analyzed via flow cytometer, immunofluorescence, and SeahorseXF platform. Lipidomics analysis demonstrated that SCFAs significantly remodeled the brain lipidome in vivo and in vitro. The most notable remodulation was observed in the metabolism of phosphatidylethanolamine plasmalogens, and mitochondrial lipids carnitine and cardiolipin. Increased mitochondrial mass, fragmentation, and hyperfusion occurred concomitant with the altered mitochondrial lipid metabolism resulting in decreased neuronal cell respiration, adenosine triphosphate (ATP) production, and increased cell death. This suggests SCFAs at levels observed in human systemic circulation can adversely alter the brain lipidome and neuronal cell function potentially negatively impacting brain health outcomes.

Dense waters (DW) formation in shelf areas and their cascading off the shelf break play a major role in ventilating deep waters, thus potentially affecting ecosystem functioning and biogeochemical cycles. However, whether DW flow across shelves may affect the composition and structure of plankton communities down to the seafloor and the particles transport over long distances has not been fully investigated. Following the 2012 north Adriatic Sea cold outbreak, DW masses were intercepted at ca. 460 km south the area of origin and compared to resident ones in term of plankton biomass partitioning (pico to micro size) and phytoplankton species composition. Results indicated a relatively higher contribution of heterotrophs in DW than in deep resident water masses, probably as result of DW-mediated advection of fresh organic matter available to consumers. DWs showed unusual high abundances of Skeletonema sp., a diatom that bloomed in the north Adriatic during DW formation. The Lagrangian numerical model set up on this diatom confirmed that DW flow could be an important mechanism for plankton/particles export to deep waters. We conclude that the predicted climate-induced variability in DW formation events could have the potential to affect the ecosystem functioning of the deeper part of the Mediterranean basin, even at significant distance from generation sites.

Many emerging arboviruses of global public health importance, such as dengue virus (DENV) and yellow fever virus (YFV), originated in sylvatic transmission cycles involving wild animals and forest-dwelling mosquitoes. Arbovirus emergence in the human population typically results from spillover transmission via bridge vectors, which are competent mosquitoes feeding on both humans and wild animals. Another related, but less studied concern, is the risk of ‘spillback’ transmission from humans into novel sylvatic cycles. We colonized a sylvatic population of Aedes malayensis from a forested area of the Nakai district in Laos to evaluate its potential as an arbovirus bridge vector. We found that this Ae. malayensis population was overall less competent for DENV and YFV than an urban population of Aedes aegypti. Olfactometer experiments showed that our Ae. malayensis colony did not display any detectable attraction to human scent in laboratory conditions. The relatively modest vector competence for DENV and YFV, combined with a lack of detectable attraction to human odor, indicate a low potential for this sylvatic Ae. malayensis population to act as an arbovirus bridge vector. However, we caution that opportunistic blood feeding on humans by sylvatic Ae. malayensis may occasionally contribute to bridge sylvatic and human transmission cycles.

Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Our data suggest that lysosomal exocytosis under doxorubicin treatment is important for cell survival and that inhibition of HDAC10 further induces DNA double-strand breaks (DSBs), providing additional mechanisms that sensitize neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.

Computer-Aided Nodule Assessment and Risk Yield (CANARY) is quantitative imaging analysis software that predicts the histopathological classification and post-treatment disease-free survival of patients with adenocarcinoma of the lung. CANARY characterizes nodules by the distribution of nine color-coded texture-based exemplars. We hypothesize that quantitative computed tomography (CT) analysis of the tumor and tumor-free surrounding lung facilitates non-invasive identification of clinically-relevant mutations in lung adenocarcinoma. Comprehensive analysis of targetable mutations (50-gene-panel) and CANARY analysis of the preoperative (≤3 months) high resolution CT (HRCT) was performed for 118 pulmonary nodules of the adenocarcinoma spectrum surgically resected between 2006–2010. Logistic regression with stepwise variable selection was used to determine predictors of mutations. We identified 140 mutations in 106 of 118 nodules. TP53 (n = 48), KRAS (n = 47) and EGFR (n = 15) were the most prevalent. The combination of Y (Yellow) and G (Green) exemplars, fibrosis within the surrounding lung and smoking status were the best discriminators for an EGFR mutation (AUC 0.77 and 0.87, respectively). None of the EGFR mutants expressing TP53 (n = 5) had a good prognosis based on CANARY features. No quantitative features were significantly associated with KRAS mutations. Our exploratory analysis indicates that quantitative CT analysis of a nodule and surrounding lung may noninvasively predict the presence of EGFR mutations in pulmonary nodules of the adenocarcinoma spectrum.

International audience; Reverse genetics is a critical tool to decrypt the biological properties of arboviruses. However, whilst reverse genetics methods have been usually applied to vertebrate cells, their use in insect cells remains uncommon due to the conjunction of laborious molecular biology techniques and of specific difficulties surrounding the transfection of such cells. To leverage reverse genetics studies in both vertebrate and mosquito cells, we designed an improved DNA transfection protocol for insect cells and then demonstrated that the simple and flexible ISA (Infectious Subgenomic Amplicons) reverse-genetics method can be efficiently applied to both mammalian and mosquito cells to generate in days recombinant infectious positive-stranded RNA viruses belonging to genera Flavivirus (Japanese encephalitis, Yellow fever, West Nile and Zika viruses) and Alphavirus (Chikungunya virus). This method represents an effective option to potentially overcome technological issues related to the study of arboviruses. Arboviruses (Arthropod-borne viruses) constitute a large group of viruses carried and spread by blood feeding arthropods, especially mosquitoes, ticks and sandflies. They can be transmitted to a variety of vertebrates and are responsible for significant morbidity and mortality amongst humans and farmed animals globally. Arboviral diseases in humans range from mild febrile illness to severe encephalitis or haemorrhagic fever 1. Iterative outbreaks worldwide over the past decades have highlighted the emergence or re-emergence potential of arboviruses, which are thus considered to be significant public and animal health threats 1–3. Most arboviruses of public health importance are single-stranded RNA viruses belonging to the families Flavi-, Toga-, or Bunyaviridae. Research focusing on arboviruses knew dramatic progress thanks to the use of reverse genetics systems allowing the study of virus life cycles, understanding the effect of specific mutations on viral replication or pathogen-esis, and designing new vaccine strategies 4,5. However, these reverse genetics systems focused to date almost exclusively on mammalian cells. Since arboviruses life cycle involves replication in both invertebrate vectors and vertebrate hosts, a simple and universal reverse genetics method allowing producing recombinant arboviruses in both vertebrate and arthropod cells would obviously facilitate the study of arbovirus biological properties, of their genomic evolution or cell interactions and restrictions. This awaited knowledge could provide in the future the key elements needed to predict outbreaks and to find efficient therapy. Unfortunately, although most reverse genetics systems proved to be efficient to recover arboviruses from vertebrate cell lines (for reviews see 4,5), very few studies have reported such systems for arthropod cells and especially for cells from Aedes mosquitoes , one of the most important arbovirus vectors globally 6. Indeed, reverse genetics systems designed for positive-sense single-stranded RNA viruses in Aedes mosquito cells are typically based to date on the lipofection or electroporation of synthetic capped RNA transcripts generated by in vitro transcription from SP6-7–9 or T7 promoter-driven 10–16 full-length viral cDNA constructs. A second system only used marginally and based on the direct transfection of a T7 promoter-driven infectious clone in an Aedes mosquito cell line stably expressing the T7 RNA polymerase was established to produce a minireplicon of the Bunyamwera negative-strand RNA virus 17. Nevertheless, these reverse genetics systems suffer from two main limitations. First, the construction of full-length viral cDNA clones remains difficult and time consuming. To circumvent this issue, we recently developed a novel bacterium-free method of reverse genetics called ISA (Infectious Subgenomic Amplicons) 18. The

In this paper, we present a spatio-temporal mathematical model for simulating the formation and growth of a thrombus. Blood is treated as a multi-constituent mixture comprised of a linear fluid phase and a thrombus (solid) phase. The transport and reactions of 10 chemical and biological species are incorporated using a system of coupled convection-reaction-diffusion (CRD) equations to represent three processes in thrombus formation: initiation, propagation and stabilization. Computational fluid dynamic (CFD) simulations using the libraries of OpenFOAM were performed for two illustrative benchmark problems: in vivo thrombus growth in an injured blood vessel and in vitro thrombus deposition in micro-channels (1.5mm x 1.6mm x 0.1mm) with small crevices (125{\mu}m x 75{\mu}m and 125{\mu}m x 137{\mu}m). For both problems, the simulated thrombus deposition agreed very well with experimental observations, both spatially and temporally. Based on the success with these two benchmark problems, which have very different flow conditions and biological environments, we believe that the current model will provide useful insight into the genesis of thrombosis in blood-wetted devices, and provide a tool for the design of less thrombogenic devices., Comment: Submission planned to Biophysical Journal

Continuous flow ventricular assist devices (cfVADs) provide a life-saving therapy for severe heart failure. However, in recent years, the incidence of device-related thrombosis (resulting in stroke, device-exchange surgery or premature death) has been increasing dramatically, which has alarmed both the medical community and the FDA. The objective of this study was to gain improved understanding of the initiation and progression of thrombosis in one of the most commonly used cfVADs, the Thoratec HeartMate II. A computational fluid dynamics simulation (CFD) was performed using our recently updated mathematical model of thrombosis. The patterns of deposition predicted by simulation agreed well with clinical observations. Furthermore, thrombus accumulation was found to increase with decreased flow rate, and can be completely suppressed by the application of anticoagulants and/or improvement of surface chemistry. To our knowledge, this is the first simulation to explicitly model the processes of platelet deposition and thrombus growth in a continuous flow blood pump and thereby replicate patterns of deposition observed clinically. The use of this simulation tool over a range of hemodynamic, hematological, and anticoagulation conditions could assist physicians to personalize clinical management to mitigate the risk of thrombosis. It may also contribute to the design of future VADs that are less thrombogenic.

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%) and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1–9 of SND1 and exons 2 to 3′ end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy.