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Background Suicide gene therapy for malignant gliomas has shown encouraging results in the latest clinical trials. However, prodrug application was most often restricted to short-term treatment (14 days), especially when replication-defective vectors were used. We previously showed that a substantial fraction of herpes simplex virus thymidine kinase (HSV-TK) transduced tumor cells survive ganciclovir (GCV) treatment in an orthotopic glioblastoma (GBM) xenograft model. Here we analyzed whether these TK+ tumor cells are still sensitive to prodrug treatment and whether prolonged prodrug treatment can enhance treatment efficacy. Methods Glioma cells positive for TK and green fluorescent protein (GFP) were sorted from xenograft tumors recurring after suicide gene therapy, and their sensitivity to GCV was tested in vitro. GBM xenografts were treated with HSV-TK/GCV, HSV-TK/valganciclovir (valGCV), or HSV-TK/valGCV + erlotinib. Tumor growth was analyzed by MRI, and survival as well as morphological and molecular changes were assessed. Results TK-GFP+ tumor cells from recurrent xenograft tumors retained sensitivity to GCV in vitro. Importantly, a prolonged period (3 mo) of prodrug administration with valganciclovir (valGCV) resulted in a significant survival advantage compared with short-term (3 wk) application of GCV. Recurrent tumors from the treatment groups were more invasive and less angiogenic compared with primary tumors and showed significant upregulation of epidermal growth factor receptor (EGFR) expression. However, double treatment with the EGFR inhibitor erlotinib did not increase therapeutic efficacy. Conclusion Long-term treatment with valGCV should be considered as a replacement for short-term treatment with GCV in clinical trials of HSV-TK mediated suicide gene therapy.

While the central nervous system is considered an immunoprivileged site and brain tumors display immunosuppressive features, both innate and adaptive immune responses affect glioblastoma (GBM) growth and treatment resistance. However, the impact of the major immune cell population in gliomas, represented by glioma-associated microglia/macrophages (GAMs), on patients' clinical course is still unclear. Thus, we aimed at assessing the immunohistochemical expression of selected microglia and macrophage markers in 344 gliomas (including gliomas from WHO grade I-IV). Furthermore, we analyzed a cohort of 241 IDH1R132H-non-mutant GBM patients for association of GAM subtypes and patient overall survival. Phenotypical properties of GAMs, isolated from high-grade astrocytomas by CD11b-based magnetic cell sorting, were analyzed by immunocytochemistry, mRNA microarray, qRT-PCR and bioinformatic analyses. A higher amount of CD68-, CD163- and CD206-positive GAMs in the vital tumor core was associated with beneficial patient survival. The mRNA expression profile of GAMs displayed an upregulation of factors that are considered as pro-inflammatory M1 (eg, CCL2, CCL3L3, CCL4, PTGS2) and anti-inflammatory M2 polarization markers (eg, MRC1, LGMN, CD163, IL10, MSR1), the latter rather being associated with phagocytic functions in the GBM microenvironment. In summary, we present evidence that human GBMs contain mixed M1/M2-like polarized GAMs and that the levels of different GAM subpopulations in the tumor core are positively associated with overall survival of patients with IDH1R132H-non-mutant GBMs.

The infiltrative nature of Glioblastoma (GBM), the most aggressive primary brain tumor, critically prevents complete surgical resection and masks tumor cells behind the blood brain barrier reducing the efficacy of systemic treatment. Here, we use a genome-wide interference screen to determine invasion-essential genes and identify the AN1/A20 zinc finger domain containing protein 3 (ZFAND3) as a crucial driver of GBM invasion. Using patient-derived cellular models, we show that loss of ZFAND3 hampers the invasive capacity of GBM, whereas ZFAND3 overexpression increases motility in cells that were initially not invasive. At the mechanistic level, we find that ZFAND3 activity requires nuclear localization and integral zinc-finger domains. Our findings indicate that ZFAND3 acts within a nuclear protein complex to activate gene transcription and regulates the promoter of invasion-related genes such as COL6A2, FN1, and NRCAM. Further investigation in ZFAND3 function in GBM and other invasive cancers is warranted., Glioblastomas (GBMs) are highly invasive brain tumours, but the underlying mechanisms of GBM invasion are unclear. Here, the authors perform an RNA interference screen and identify AN1-Type Zinc Finger protein 3 (ZFAND3) as a regulator of GBM invasion, and find that it acts through the transcriptional regulation of invasion-related genes.

Patient-derived cancer models are essential tools for studying tumor biology and preclinical interventions. Here, we show that glioma patient-derived orthotopic xenografts (PDOXs) enable long-term propagation of patient tumors and represent clinically relevant patient avatars. We created a large collection of PDOXs from primary and recurrent gliomas with and without mutations in IDH1, which retained histopathological, genetic, epigenetic and transcriptomic features of patient tumors with no mouse-specific clonal evolution. Longitudinal PDOX models recapitulate the limited genetic evolution of gliomas observed in patient tumors following treatment. PDOX-derived standardized tumor organoid cultures enabled assessment of drug responses, which were validated in mice. PDOXs showed clinically relevant responses to Temozolomide and to targeted treatments such as EGFR and CDK4/6 inhibitors in (epi)genetically defined groups, according to MGMT promoter and EGFR/CDK status respectively. Dianhydrogalactitol, a bifunctional alkylating agent, showed promising potential against glioblastoma. Our study underlines the clinical relevance of glioma PDOX models for translational research and personalized treatment studies.

Simple Summary Glioblastoma (GBM) is the most aggressive brain tumor and most resistant to therapy. The identification of novel predictive biomarkers or targets to counteract chemoresistance, requires a better understanding of the GBM primary response to therapy. The aim of our study was to assess the molecular response of GBM to the standard of care chemotherapy by temozolomide (TMZ). We established a comprehensive map of gene expression changes after treatment and discovered that GBM cells elicit a coordinated gene expression program after chemotherapy that differs between sensitive and resistant cells. We found that a novel class of genes expressed as long non-coding RNAs (lncRNAs) is involved in gene regulatory circuits in GBM and could represent novel markers of GBM patient prognosis. By shedding light on the involvement of the non-coding genome in GBM, our results may provide new mechanistic insight on lncRNAs and their importance in chemoresistance. Abstract Resistance to chemotherapy by temozolomide (TMZ) is a major cause of glioblastoma (GBM) recurrence. So far, attempts to characterize factors that contribute to TMZ sensitivity have largely focused on protein-coding genes, and failed to provide effective therapeutic targets. Long noncoding RNAs (lncRNAs) are essential regulators of epigenetic-driven cell diversification, yet, their contribution to the transcriptional response to drugs is less understood. Here, we performed RNA-seq and small RNA-seq to provide a comprehensive map of transcriptome regulation upon TMZ in patient-derived GBM stem-like cells displaying different drug sensitivity. In a search for regulatory mechanisms, we integrated thousands of molecular associations stored in public databases to generate a background “RNA interactome”. Our systems-level analysis uncovered a coordinated program of TMZ response reflected by regulatory circuits that involve transcription factors, mRNAs, miRNAs, and lncRNAs. We discovered 22 lncRNAs involved in regulatory loops and/or with functional relevance in drug response and prognostic value in gliomas. Thus, the investigation of TMZ-induced gene networks highlights novel RNA-based predictors of chemosensitivity in GBM. The computational modeling used to identify regulatory circuits underlying drug response and prioritizing gene candidates for functional validation is applicable to other datasets.

Next-generation sequencing (NGS) analyses on DNA derived from archived Formalin-Fixed Paraffin-Embedded (FFPE) clinical material can provide a powerful tool in oncology research and clinical diagnostics. Although several studies have established that NGS can be performed using DNA from FFPE tissue, the accuracy and reproducibility of such analyses, as well as their robustness to the biomolecular quality of the samples used, remains a matter of debate. Excellent reviews have recently been published, providing evidence-based best practices for FFPE DNA extraction. Alternative fixatives exist, although their implementation in clinical practice is difficult. In this article, we present (i) a review of fixed tissue DNA preanalytics with a special focus on DNA extraction and fixed tissue sample qualification and (ii) results from comparisons between different methods of DNA extraction from tissue samples that have been fixed or stabilized by different methods, in terms of NGS metrics and different DNA quality metrics.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.

BACKGROUND Percutaneous cholecystostomy (PC) is an alternative to cholecystectomy for acute calculous cholecystitis (ACC) in patients with high mortality risk, but the catheters' and patients' management remains unclear. This study aimed to determine outcomes after PC and to define surgical strategy. METHODS All patients who underwent PC between 2009 and 2014 for ACC in a single institution were reviewed for outcomes and postdrainage management. RESULTS Forty-one patients underwent PC with a median age of 77 years. Twenty patients (45%) presented American Society of Anesthesiologists (ASA) score 3-4, and all had cholecystitis grade II or III. The 6-month mortality was 17% (7/41 patients). Twelve patients (29%) presented PC-related complications. Catheters were removed, clamped, left open, and dislodged for 17, 9, 5, and 5 patients, respectively. Three patients died after PC, and data were missing for 2 patients. Twenty-five (61%) patients underwent cholecystectomy after PC, and were significantly younger (median age, 70 vs. 83; P

Glioblastoma (GBM), the most frequent and aggressive malignant primary brain tumor, is characterized by a highly invasive growth. In our previous study we showed that overexpression of Carboxypeptidase E (CPE) mitigated glioma cell migration. In the present study we aimed at deciphering the regulatory mechanisms of the secreted form of CPE (sCPE). By transcriptome analysis and inhibition of signaling pathways involved in the regulation of cell growth and motility, we discovered that overexpression of sCPE was accompanied by differential regulation of mRNAs connected to the motility-associated networks, among others FAK, PAK, Cdc42, integrin, STAT3 as well as TGF-β. Especially SLUG was downregulated in sCPE-overexpressing glioma cells, paralleled by reduced expression of matrix-metalloproteinases (MMP) and, in consequence, by decreased cell migration. Expression of SLUG was regulated by ERK since inhibition of ERK reverted sCPE-mediated SLUG downregulation and enhanced cell motility. In a mouse glioma model, overexpression of sCPE significantly prolonged survival. Our results implicate a novel role for sCPE that mainly affects the expression of motility-associated genes via several signal pathways.

Background The amount of publicly available cancer-related “omics” data is constantly growing and can potentially be used to gain insights into the tumour biology of new cancer patients, their diagnosis and suitable treatment options. However, the integration of different datasets is not straightforward and requires specialized approaches to deal with heterogeneity at technical and biological levels. Methods Here we present a method that can overcome technical biases, predict clinically relevant outcomes and identify tumour-related biological processes in patients using previously collected large discovery datasets. The approach is based on independent component analysis (ICA) – an unsupervised method of signal deconvolution. We developed parallel consensus ICA that robustly decomposes transcriptomics datasets into expression profiles with minimal mutual dependency. Results By applying the method to a small cohort of primary melanoma and control samples combined with a large discovery melanoma dataset, we demonstrate that our method distinguishes cell-type specific signals from technical biases and allows to predict clinically relevant patient characteristics. We showed the potential of the method to predict cancer subtypes and estimate the activity of key tumour-related processes such as immune response, angiogenesis and cell proliferation. ICA-based risk score was proposed and its connection to patient survival was validated with an independent cohort of patients. Additionally, through integration of components identified for mRNA and miRNA data, the proposed method helped deducing biological functions of miRNAs, which would otherwise not be possible. Conclusions We present a method that can be used to map new transcriptomic data from cancer patient samples onto large discovery datasets. The method corrects technical biases, helps characterizing activity of biological processes or cell types in the new samples and provides the prognosis of patient survival.

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment., Cancer stem cells (CSCs) comprise a putative population that can drive growth and resistance. Here, in glioblastoma models the authors show that rather than being a distinct clonal entity, the CSC population represents a plastic state adoptable by most cancer cells via reversible state transitions induced by the microenvironment.

Background: The topological analysis of networks extracted from different types of “omics” data is a useful strategy for characterizing biologically meaningful properties of the complex systems underlying these networks. In particular, the biological significance of highly connected genes in diverse molecular networks has been previously determined using data from several model organisms and phenotypes. Despite such insights, the predictive potential of candidate hubs in gene co-expression networks in the specific context of cancer-related drug experiments remains to be deeply investigated. The examination of such associations may offer opportunities for the accurate prediction of anticancer drug responses. Methods: Here, we address this problem by: a) analyzing a co-expression network obtained from thousands of cancer cell lines, b) detecting significant network hubs, and c) assessing their capacity to predict drug sensitivity using data from thousands of drug experiments. We investigated the prediction capability of those genes using a multiple linear regression model, independent datasets, comparisons with other models and our own in vitro experiments. Results: These analyses led to the identification of 47 hub genes, which are implicated in a diverse range of cancer-relevant processes and pathways. Overall, encouraging agreements between predicted and observed drug sensitivities were observed in public datasets, as well as in our in vitro validations for four glioblastoma cell lines and four drugs. To facilitate further research, we share our hub-based drug sensitivity prediction model as an online tool. Conclusions: Our research shows that co-expression network hubs are biologically interesting and exhibit potential for predicting drug responses in vitro. These findings motivate further investigations about the relevance and application of our unbiased discovery approach in pre-clinical, translationally-oriented research.

Deregulated cell migration and invasion are hallmarks of metastatic cancer cells. Phosphorylation on residue Ser5 of the actin-bundling protein L-plastin activates L-plastin and has been reported to be crucial for invasion and metastasis. Here, we investigate signal transduction leading to L-plastin Ser5 phosphorylation using 4 human breast cancer cell lines. Whole-genome microarray analysis comparing cell lines with different invasive capacities and corresponding variations in L-plastin Ser5 phosphorylation level revealed that genes of the ERK/MAPK pathway are differentially expressed. It is noteworthy that in vitro kinase assays showed that ERK/MAPK pathway downstream ribosomal protein S6 kinases α-1 (RSK1) and α-3 (RSK2) are able to directly phosphorylate L-plastin on Ser5. Small interfering RNA- or short hairpin RNA-mediated knockdown and activation/inhibition studies followed by immunoblot analysis and computational modeling confirmed that ribosomal S6 kinase (RSK) is an essential activator of L-plastin. Migration and invasion assays showed that RSK knockdown led to a decrease of up to 30% of migration and invasion of MDA-MB-435S cells. Although the presence of L-plastin was not necessary for migration/invasion of these cells, immunofluorescence assays illustrated RSK-dependent recruitment of Ser5-phosphorylated L-plastin to migratory structures. Altogether, we provide evidence that the ERK/MAPK pathway is involved in L-plastin Ser5 phosphorylation in breast cancer cells with RSK1 and RSK2 kinases able to directly phosphorylate L-plastin residue Ser5.

Cancer is driven by genomic alterations, but the processes causing this disease are largely performed by proteins. However, proteins are harder and more expensive to measure than genes and transcripts. To catalyze developments of methods to infer protein levels from other omics measurements, we leveraged crowdsourcing via the NCI-CPTAC DREAM proteogenomic challenge. We asked for methods to predict protein and phosphorylation levels from genomic and transcriptomic data in cancer patients. The best performance was achieved by an ensemble of models, including as predictors transcript level of the corresponding genes, interaction between genes, conservation across tumor types, and phosphosite proximity for phosphorylation prediction. Proteins from metabolic pathways and complexes were the best and worst predicted, respectively. The performance of even the best-performing model was modest, suggesting that many proteins are strongly regulated through translational control and degradation. Our results set a reference for the limitations of computational inference in proteogenomics. A record of this paper's transparent peer review process is included in the Supplemental Information.

Biliary leak following severe blunt liver injuries is a complex problem becoming more frequent with improvements in non-operative management. Standard treatment requires main bile duct drainage usually performed by endoscopic sphincterotomy and stent placement. We report our experience with cholecystostomy as a first minimally invasive diagnostic and therapeutic approach. We performed a retrospective analysis of consecutive patients with post-traumatic biliary leak between 2006 and 2015. In the first period (2006–2010), biliary fistula was managed using perihepatic drainage and endoscopic, percutaneous or surgical main bile duct drainage. After 2010, cholecystostomy as an initial minimally invasive approach was performed. Of 341 patients with blunt liver injury, 18 had a post-traumatic biliary leak. Ten patients received standard treatment and eight patients underwent cholecystostomy. The cholecystostomy (62.5%) and the standard treatment (80%) groups presented similar success rates as the first biliary drainage procedure (p = 0.41). Cholecystostomy presented no severe complications and resulted, when successful, in a bile flow rate inversion between the perihepatic drains and the gallbladder drain within a median [IQR] 4 days [1–7]. The median time for bile leak resolution was 26 days in the cholecystostomy group and 39 days in the standard treatment group (p = 0.09). No significant difference was found considering median duration of hospital stay (54 and 74 days, respectively, p = 0.37) or resuscitation stay (17.5 and 19.5 days, p = 0.59). Cholecystostomy in non-operative management of biliary fistula after blunt liver injury could be an effective, simple and safe first-line procedure in the diagnostic and therapeutic approach of post-traumatic biliary tract injuries.

We report that CD47 was upregulated in different EMT-activated human breast cancer cells versus epithelial MCF7 cells. Overexpression of SNAI1 or ZEB1 in epithelial MCF7 cells activated EMT and upregulated CD47 while siRNA-mediated targeting of SNAI1 or ZEB1 in mesenchymal MDA-MB-231 cells reversed EMT and strongly decreased CD47. Mechanistically, SNAI1 and ZEB1 upregulated CD47 by binding directly to E-boxes in the human CD47 promoter. TCGA and METABRIC data sets from breast cancer patients revealed that CD47 correlated with SNAI1 and Vimentin. At functional level, different EMT-activated breast cancer cells were less efficiently phagocytosed by macrophages vs. MCF7 cells. The phagocytosis of EMT-activated cells was rescued by using CD47 blocking antibody or by genetic targeting of SNAI1, ZEB1 or CD47. These results provide a rationale for an innovative preclinical combination immunotherapy based on PD-1/PD-L1 and CD47 blockade along with EMT inhibitors in patients with highly aggressive, mesenchymal, and metastatic breast cancer.

Background One of the main current challenges in computational biology is to make sense of the huge amounts of multidimensional experimental data that are being produced. For instance, large cohorts of patients are often screened using different high-throughput technologies, effectively producing multiple patient-specific molecular profiles for hundreds or thousands of patients. Results We propose and implement a network-based method that integrates such patient omics data into Patient Similarity Networks. Topological features derived from these networks were then used to predict relevant clinical features. As part of the 2017 CAMDA challenge, we have successfully applied this strategy to a neuroblastoma dataset, consisting of genomic and transcriptomic data. In particular, we observe that models built on our network-based approach perform at least as well as state of the art models. We furthermore explore the effectiveness of various topological features and observe, for instance, that redundant centrality metrics can be combined to build more powerful models. Conclusion We demonstrate that the networks inferred from omics data contain clinically relevant information and that patient clinical outcomes can be predicted using only network topological data. Reviewers This article was reviewed by Yang-Yu Liu, Tomislav Smuc and Isabel Nepomuceno. Electronic supplementary material The online version of this article (10.1186/s13062-018-0214-9) contains supplementary material, which is available to authorized users.

International audience; Background Islet transplantation is indicated for patients with type 1 diabetes with severe hypoglycaemia or after kidney transplantation. We did a randomised trial to assess the efficacy and safety of islet transplantation compared with insulin therapy in these patients. Methods In this multicentre, open-label, randomised controlled trial, we randomly assigned (1:1) patients with type 1 diabetes at 15 university hospitals to receive immediate islet transplantation or intensive insulin therapy (followed by delayed islet transplantation). Eligible patients were aged 18-65 years and had severe hypoglycaemia or hypoglycaemia unawareness, or kidney grafts with poor glycaemic control. We used computer-generated randomisation, stratified by centre and type of patient. Islet recipients were scheduled to receive 11000 islet equivalents per kg bodyweight in one to three infusions. The primary outcome was proportion of patients with a modified (beta-score (in which an overall score of 0 was not allocated when stimulated C-peptide was negative) of 6 or higher at 6 months after first islet infusion in the immediate transplantation group or 6 months after randomisation in the insulin group. The primary analysis included all patients who received the allocated intervention; safety was assessed in all patients who received islet infusions. This trial is registered with ClinicalTrials.gov, number NCT01148680, and is completed. Findings Between July 8, 2010, and July 29, 2013, 50 patients were randomly assigned to immediate islet transplantation (n=26) or insulin treatment (n=24), of whom three (one in the immediate islet transplantation group and two in the insulin therapy group) did not receive the allocated intervention. Median follow-up was 184 days (IQR 181-186) in the immediate transplantation group and 185 days (172-201) in the insulin therapy group. At 6 months, 16 (64% [95% CI 43-82]) of 25 patients in the immediate islet transplantation group had a modified (beta-score of 6 or higher versus none (0% [0-15]) of the 22 patients in the insulin group (p\textless0.0001). At 12 months after first infusion, bleeding complications had occurred in four (7% [2-18]) of 55 infusions, and a decrease in median glomerular filtration rate from 90-5 mL/min (IQR 76-6-94-0) to 71-8 mL/min (59-0-89-0) was observed in islet recipients who had not previously received a kidney graft and from 63 .0 mL/min (55.0-71-0) to 57. 0 mL/min (45-5-65 . 1) in islet recipients who had previously received a kidney graft. Interpretation For the indications assessed in this study, islet transplantation effectively improves metabolic outcomes. Although studies with longer-term follow-up are needed, islet transplantation seems to be a valid option for patients with severe, unstable type 1 diabetes who are not responding to intensive medical treatments. However, immunosuppression can affect kidney function, necessitating careful selection of patients. Copyright (C) 2018 Elsevier Ltd. All rights reserved.

Background RNA sequencing (RNA-seq) and microarrays are two transcriptomics techniques aimed at the quantification of transcribed genes and their isoforms. Here we compare the latest Affymetrix HTA 2.0 microarray with Illumina 2000 RNA-seq for the analysis of patient samples - normal lung epithelium tissue and squamous cell carcinoma lung tumours. Protein coding mRNAs and long non-coding RNAs (lncRNAs) were included in the study. Results Both platforms performed equally well for protein-coding RNAs, however the stochastic variability was higher for the sequencing data than for microarrays. This reduced the number of differentially expressed genes and genes with predictive potential for RNA-seq compared to microarray data. Analysis of this variability revealed a lack of reads for short and low abundant genes; lncRNAs, being shorter and less abundant RNAs, were found especially susceptible to this issue. A major difference between the two platforms was uncovered by analysis of alternatively spliced genes. Investigation of differential exon abundance showed insufficient reads for many exons and exon junctions in RNA-seq while the detection on the array platform was more stable. Nevertheless, we identified 207 genes which undergo alternative splicing and were consistently detected by both techniques. Conclusions Despite the fact that the results of gene expression analysis were highly consistent between Human Transcriptome Arrays and RNA-seq platforms, the analysis of alternative splicing produced discordant results. We concluded that modern microarrays can still outperform sequencing for standard analysis of gene expression in terms of reproducibility and cost. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3819-y) contains supplementary material, which is available to authorized users.

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.

Extracorporeal high-intensity focused ultrasound (HIFU) is a minimally invasive therapy considered with increased interest for the ablation of small tumors in deeply located organs while sparing surrounding critical tissues. A multitude of preclinical and clinical studies have showed the feasibility of the method; however, concurrently they showed several obstacles, among which the management of respiratory motion of abdominal organs is at the forefront. The aim of this review is to describe the different methods that have been proposed for managing respiratory motion and to identify their advantages and weaknesses. First, we specify the characteristics of respiratory motion for the liver, kidneys, and pancreas and the problems it causes during HIFU planning, treatment, and monitoring. Second, we make an inventory of the preclinical and clinical approaches used to overcome the problem of organ motion. Third, we analyze their respective benefits and drawbacks to identify the remaining physical, technological, and clinical challenges. We thereby consider the outlook of motion compensation techniques and those that would be the most suitable for clinical use, particularly under magnetic resonance thermometry monitoring.

The treatment of liver cancer is a major public health issue because the liver is a frequent site for both primary and secondary tumors. Rib heating represents a major obstacle for the application of extracorporeal focused ultrasound to liver ablation. Magnetic resonance (MR)-guided external shielding of acoustic obstacles (eg, the ribs) was investigated here to avoid unwanted prefocal energy deposition in the pathway of the focused ultrasound beam.Ex vivo and in vivo (7 female sheep) experiments were performed in this study. Magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU) was performed using a randomized 256-element phased-array transducer (f∼1 MHz) and a 3-T whole-body clinical MR scanner. A physical mask was inserted in the prefocal beam pathway, external to the body, to block the energy normally targeted on the ribs. The effectiveness of the reflecting material was investigated by characterizing the efficacy of high-intensity focused ultrasound beam reflection and scattering on its surface using Schlieren interferometry. Before high-intensity focused ultrasound sonication, the alignment of the protectors with the conical projections of the ribs was required and achieved in multiple steps using the embedded graphical tools of the MR scanner. Multiplanar near real-time MR thermometry (proton resonance frequency shift method) enabled the simultaneous visualization of the local temperature increase at the focal point and around the exposed ribs. The beam defocusing due to the shielding was evaluated from the MR acoustic radiation force impulse imaging data.Both MR thermometry (performed with hard absorber positioned behind a full-aperture blocking shield) and Schlieren interferometry indicated a very good energy barrier of the shielding material. The specific temperature contrast between rib surface (spatial average) and focus, calculated at the end point of the MRgHIFU sonication, with protectors vs no protectors, indicated an important reduction of the temperature elevation at the ribs' surface, typically by 3.3 ± 0.4 in vivo. This was translated into an exponential reduction in thermal dose by several orders of magnitude. The external shielding covering the full conical shadow of the ribs was more effective when the protectors could be placed close to the ribs' surface and had a tendency to lose its efficiency when placed further from the ribs. Hepatic parenchyma was safely ablated in vivo using this rib-sparing strategy and single-focus independent sonications.A readily available, MR-compatible, effective, and cost-competitive method for rib protection in transcostal MRgHIFU was validated in this study, using specific reflective strips. The current approach permitted safe intercostal ablation of small volumes (0.7 mL) of liver parenchyma.

Transcriptional profiling was performed on 452 RNA preparations isolated from various types of pancreatic tissue from tumour patients and healthy donors, with a particular focus on peritumoral samples. Pancreatic ductal adenocarcinomas (PDAC) and cystic tumours were most different in these non‐tumorous tissues surrounding them, whereas the actual tumours exhibited rather similar transcript patterns. The environment of cystic tumours was transcriptionally nearly identical to normal pancreas tissue. In contrast, the tissue around PDAC behaved a lot like the tumour, indicating some kind of field defect, while showing far less molecular resemblance to both chronic pancreatitis and healthy tissue. This suggests that the major pathogenic difference between cystic and ductal tumours may be due to their cellular environment rather than the few variations between the tumours. Lack of correlation between DNA methylation and transcript levels makes it unlikely that the observed field defect in the peritumoral tissue of PDAC is controlled to a large extent by such epigenetic regulation. Functionally, a strikingly large number of autophagy‐related transcripts was changed in both PDAC and its peritumoral tissue, but not in other pancreatic tumours. A transcription signature of 15 autophagy‐related genes was established that permits a prognosis of survival with high accuracy and indicates the role of autophagy in tumour biology., What's new? To date, relatively few RNA expression data are available on pancreatic tumour types other than pancreatic ductal adenocarcinoma (PDAC) or peritumoral tissues. Here, the authors found that transcriptionally, peritumoral tissues of PDAC behave much like the tumours, although resembling normal tissue in cell composition. In contrast, transcription in the environment of cystic pancreatic tumours is basically identical to that of normal tissue, suggesting an effect of peritumoral tissues on the strong pathogenic difference between PDAC and cystic tumours. At the functional level, autophagy‐related genes are uniquely changed in PDAC and its peritumoral tissue, with their expression variation predicting patient survival.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca(2+)-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred µm, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration. We thank C. Hoffmann and J. Esposito for technical support, A. Feenstra (VUA) for co-supervision of N.K., and R. Schneider (LCSB) for co-supervision of G.A. during her work at LIH and current support at LCSB. This research was funded by Luxembourg's National Research Fund (FNR) and the Swiss National Research Foundation (SNF), INFUSED project (www.infused-project.eu). N.M. was supported by Comunidad de Madrid (Fibroteam P2010/BMD-2321) and the ERC (Starting Grant 2013 337703 zebraHeart). Zebrafish drawing in Figure 1 was adapted from Wikimedia Commons (http://bit.ly/1PL92vj). Sí

Channelrhodopsins are used to optogenetically depolarize neurons. We engineered a variant of channelrhodopsin, denoted Red-activatable Channelrhodopsin (ReaChR), that is optimally excited with orange to red light (λ ~ 590 to 630 nm) and offers improved membrane trafficking, higher photocurrents, and faster kinetics compared with existing red-shifted channelrhodopsins. Red light is more weakly scattered by tissue and absorbed less by blood than the blue to green wavelengths required by other channelrhodopsin variants. ReaChR expressed in vibrissa motor cortex was used to drive spiking and vibrissa motion in awake mice when excited with red light through intact skull. Precise vibrissa movements were evoked by expressing ReaChR in the facial motor nucleus in the brainstem and illuminating with red light through the external auditory canal. Thus, ReaChR enables transcranial optical activation of neurons in deep brain structures without the need to surgically thin the skull, form a transcranial window, or implant optical fibers.

Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions. J. Comp. Neurol. 519:2390–2416, 2011. © 2011 Wiley-Liss, Inc.

We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC) by developing sensor cells stably expressing a Ca(2+) permeable LGIC and a genetically encoded Förster (or fluorescence) resonance energy transfer (FRET)-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A) serotonin receptors and a chimera of human α7/mouse 5-HT(3A) receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

The neurovascular system may be viewed as a distributed nervous system within the brain. It transforms local neuronal activity into a change in the tone of smooth muscle that lines the walls of arterioles and microvessels. We review the current state of neurovascular coupling, with an emphasis on signaling molecules that convey information from neurons to neighboring vessels. At the level of neocortex, this coupling is mediated by: (i) a likely direct interaction with inhibitory neurons, (ii) indirect interaction, via astrocytes, with excitatory neurons, and (iii) fiber tracts from subcortical layers. Substantial evidence shows that control involves competition between signals that promote vasoconstriction versus vasodilation. Consistent with this picture is evidence that, under certain circumstances, increased neuronal activity can lead to vasoconstriction rather than vasodilation. This confounds naïve interpretations of functional brain images. We discuss experimental approaches to detect signaling molecules in vivo with the goal of formulating an empirical basis for the observed logic of neurovascular control.

Background-: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission. Results-: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice. Conclusions-: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDP-glucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for mu-opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.

Background In the post-genomic era, systems-level studies are being performed that seek to explain complex biological systems by integrating diverse resources from fields such as genomics, proteomics or transcriptomics. New information management systems are now needed for the collection, validation and analysis of the vast amount of heterogeneous data available. Multiple alignments of complete sequences provide an ideal environment for the integration of this information in the context of the protein family. Results MACSIMS is a multiple alignment-based information management program that combines the advantages of both knowledge-based and ab initio sequence analysis methods. Structural and functional information is retrieved automatically from the public databases. In the multiple alignment, homologous regions are identified and the retrieved data is evaluated and propagated from known to unknown sequences with these reliable regions. In a large-scale evaluation, the specificity of the propagated sequence features is estimated to be >99%, i.e. very few false positive predictions are made. MACSIMS is then used to characterise mutations in a test set of 100 proteins that are known to be involved in human genetic diseases. The number of sequence features associated with these proteins was increased by 60%, compared to the features available in the public databases. An XML format output file allows automatic parsing of the MACSIM results, while a graphical display using the JalView program allows manual analysis. Conclusion MACSIMS is a new information management system that incorporates detailed analyses of protein families at the structural, functional and evolutionary levels. MACSIMS thus provides a unique environment that facilitates knowledge extraction and the presentation of the most pertinent information to the biologist. A web server and the source code are available at http://bips.u-strasbg.fr/MACSIMS/.

A deterministic Markov process was developed to estimate the efficiency of a fully controlled photobioreactor by calculating the growth of the Rhodophyte Porphyridium cruentum. It was assumed that microalgal growth, in strictly controlled and non-nutrient-limited conditions, is a function of the amount of light energy received. The light sources delivered a continuous 206 microE m(-2) s(-1) average photon flux density to cultures reaching concentrations of 3 g l(-1) in batch and 0.7 g l(-1) in chemostat. The concentration time-courses calculated compared satisfactorily with measured results for both batch and continuous cultures. The quality of simulation in these two cases validated the hypotheses made, especially for the linearity of light bioconversion, and allowed the model to be used for further exploration of the photobioreactor-operating domain. Distribution of the specific growth rate as a function of time and radial position was compared for the two simulated cases. The use of an initial batch of several days prior to the dilution of a continuous culture proved theoretically beneficial for overall production. The influence of the dilution rate and of light-path length on surface productivity and concentration at steady state were demonstrated.