Academic expertise in assisting private companies in the fields of environment and environmental toxicology: the role of individual expertise.

The scientific knowledge produced by academic research can be valued in all sectors of human activity, including private sector. The ROVALTAIN Foundation organized a round-table during its scientific day in 2019. It crossed the points of view of academic scientists and industrial partners, addressing five main topics. The first one concerned the validation of a common definition of the academic research/private partners interface. Then, the group discussed the place for academic expertise in the corporate world; the advantages of involving academic researchers in expertise for the private sector; and the limits of this model. To conclude, the need of a third party, like the ROVALTAIN Foundation, as a catalyzer in building the interface between academic research and private partners has been discussed.

The contribution of genomics in the medicine of tomorrow, clinical applications and issues.

Fifteen years after the completion of first human genome sequencing, the technique is almost a commodity but there is still little evidence of its usefulness as a diagnostic, prognostic or therapeutic tool. In France, the France genomics plan 2025 was launched in 2015 with the goal of integrating genomic tests into clinical practice and developing a National genomics network including industrial partnerships. Reflection on scientific applications and operational or societal issues is needed to make recommendations to help better associate Genomics and the medicine of tomorrow. In the perspective of personalized Evidence-based Medicine, studies with an appropriate methodological level to improve the definition of evidence should be promoted. The many operational challenges require the implementation of organisations and means to streamline the process of results reporting, and regulatory adaptations concerning the status of professions involved, the management of data generated, and the consent of patients. In parallel, genetic training for healthcare professionals and raising awareness on genetic tests for the public should be considered. The ethical stake should also be taken into account, especially on the participation of the patient in decisions concerning them and integrating the notion of uncertainty into the information given. The sociological effects on the experience and expectations of patients and the general population towards genomic medicine should also be evaluated to improve information, prevention and support for people. Finally, medico-economic studies must be conducted to inform policy-makers on the cost-effectiveness of complete genome sequencing for population health.

Tracking Cancer Genetic Evolution using OncoTrack.

It is difficult for existing methods to quantify, and track the constant evolution of cancers due to high heterogeneity of mutations. However, structural variations associated with nucleotide number changes show repeatable patterns in localized regions of the genome. Here we introduce SPKMG, which generalizes nucleotide number based properties of genes, in statistical terms, at the genome-wide scale. It is measured from the normalized amount of aligned NGS reads in exonic regions of a gene. SPKMG values are calculated within OncoTrack. SPKMG values being continuous numeric variables provide a statistical metric to track DNA level changes. We show that SPKMG measures of cancer DNA show a normative pattern at the genome-wide scale. The analysis leads to the discovery of core cancer genes and also provides novel dynamic insights into the stage of cancer, including cancer development, progression, and metastasis. This technique will allow exome data to also be used for quantitative LOH/CNV analysis for tracking tumour progression and evolution with a higher efficiency.

Characterizing short stature by insulin-like growth factor axis status and genetic associations: results from the prospective, cross-sectional, epidemiogenetic EPIGROW study.

CONTEXT: Serum IGF-I levels are often low in patients with short stature (SS) without defined etiology. Hence, genetic investigations have focused on the GH-IGF-I axis. OBJECTIVE: Our objectives were to characterize IGF-I axis status and search for a broader range of genetic associations in children with SS and normal GH. DESIGN AND SETTING: We conducted a prospective, cross-sectional, epidemiogenetic case-control study in 9 European countries (2008-2010). PARTICIPANTS: Children (n = 275) aged ≥2 years with SS without defined etiology (≤-2.5 height SD score [SDS]) and ≥1 peak GH ≥7 µg/L) were recruited. METHODS: Serum IGF-I, IGF-binding protein-3 (IGFBP-3), and acid-labile subunit (ALS) levels were measured in a central laboratory. Candidate gene exome sequencing was performed in this cohort and ethnicity-matched controls. RESULTS: Serum IGF-I, IGFBP-3, and ALS levels were highly correlated, but there was a discrepancy between prevalence of IGF-I, IGFBP-3, and ALS deficiencies (53%, 30%, and 0.8%, respectively). An insertion-deletion (Indel) on the IGF1 gene (P = 1.2 × 10(-5), Bonferroni-corrected; case vs control frequency: 0.04 vs 0.112), an Indel on NFKB1 (P = 1.36 × 10(-10); case vs control frequency: 0.464 vs 0.272), and 2 single-nucleotide polymorphisms on ZBTB38 (P < 2.3 × 10(-6)) were associated with SS. At P < 10(-4), single-nucleotide polymorphisms on genes related to protein kinase regulation, MAPK, and Fanconi pathways were also associated with SS. CONCLUSIONS: IGF-I deficiency is a common feature in SS without defined etiology; an Indel in the IGF1 gene was associated with SS. However, genes involved in transcriptional regulation (NFKB1 and ZBTB38) and growth factor signaling were also associated, providing further candidates for genetic investigations on individual patients.

Molecular pathogenesis of neuroendocrine tumors: implications for current and future therapeutic approaches.

The treatment landscape and biologic understanding of neuroendocrine tumors (NET) has shifted dramatically in recent years. Recent studies have shown that somatostatin analogues have the potential not only to control symptoms of hormone hypersecretion but also have the ability to slow tumor growth in patients with advanced carcinoid. The results of clinical trials have further shown that the VEGF pathway inhibitor sunitinib and the mTOR inhibitor everolimus have efficacy in patients with advanced pancreatic NETs. The efficacy of these targeted therapies in NET suggests that the molecular characterization of NETs may provide an avenue to predict both which patients may benefit most from the treatment and to overcome potential drug resistance. Recent genomic studies of NETs have further suggested that pathways regulating chromatin remodeling and epigenetic modification may play a key role in regulating NET growth. These observations offer the potential for new therapeutic and diagnostic advances for patients with NET.

Introduction to gene therapy: a clinical aftermath.

Despite three decades of huge progress in molecular genetics, in cloning of disease causative gene as well as technology breakthroughs in viral biotechnology, out of thousands of gene therapy clinical trials that have been initiated, only very few are now reaching regulatory approval. We shall review some of the major hurdles, and based on the current either positive or negative examples, we try to initiate drawing a learning curve from experience and possibly identify the major drivers for future successful achievement of human gene therapy trials.

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis.

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle.

In-depth haplotype analysis of ABCA1 gene polymorphisms in relation to plasma ApoA1 levels and myocardial infarction.

OBJECTIVE: By regulating the cellular cholesterol efflux from peripheral cells to high-density lipoprotein, the ABCA1 protein is suspected to play a key role in lipid homeostasis and atherosclerosis. Twenty-six polymorphisms of the ABCA1 gene were genotyped and tested for association with plasma levels of ApoA1 and myocardial infarction (MI) in the ECTIM study. METHODS AND RESULTS: In addition to single-locus analysis, a systematic exploration of all possible haplotype effects was performed, with this exploration being performed on a minimal set of "tag" polymorphisms that define the haplotype structure of the gene. Two polymorphisms were associated with plasma levels of ApoA1, 1 in the promoter (C-564T) and 1 in the coding (R1587K) regions, whereas only 1 polymorphism (R219K) was associated with the risk of MI. However, no haplotype effect was detected on ApoA1 variability or on the risk of MI. CONCLUSIONS: ABCA1 gene polymorphisms but not haplotypes are involved in the variability of plasma ApoA1 and the susceptibility to coronary artery disease.

Parkin gene inactivation alters behaviour and dopamine neurotransmission in the mouse.

Mutations of the parkin gene are the most frequent cause of early onset autosomal recessive parkinsonism (EO-AR). Here we show that inactivation of the parkin gene in mice results in motor and cognitive deficits, inhibition of amphetamine-induced dopamine release and inhibition of glutamate neurotransmission. The levels of dopamine are increased in the limbic brain areas of parkin mutant mice and there is a shift towards increased metabolism of dopamine by MAO. Although there was no evidence for a reduction of nigrostriatal dopamine neurons in the parkin mutant mice, the level of dopamine transporter protein was reduced in these animals, suggesting a decreased density of dopamine terminals, or adaptative changes in the nigrostriatal dopamine system. GSH levels were increased in the striatum and fetal mesencephalic neurons from parkin mutant mice, suggesting that a compensatory mechanism may protect dopamine neurons from neuronal death. These parkin mutant mice provide a valuable tool to better understand the preclinical deficits observed in patients with PD and to characterize the mechanisms leading to the degeneration of dopamine neurons that could provide new strategies for neuroprotection.

Characterization of the ABCA transporter subfamily: identification of prokaryotic and eukaryotic members, phylogeny and topology.

An alignment of the mammalian ABCA transporters enabled the identification of sequence segments, specific to the ABCA subfamily, which were used as queries to search for eukaryotic and prokaryotic homologues. Thirty-seven eukaryotic half and full-length transporters were found, and a close relationship with prokaryotic subfamily 7 transporters was detected. Each half of the ABCA full-transporters is predicted to comprise a membrane-spanning domain (MSD) composed of six helices and a large extracellular loop, followed by a nucleotide-binding domain (NBD) and a conserved cytoplasmic 80-residue sequence, which might have a regulatory function. The topology predicted for the ABCA transporters was compared to the crystal structures of the MsbA and BtuCD bacterial transporters. The alignment of the MSD and NBD domains provided an estimate of the degree of residue conservation in the cytoplasmic, extracellular and transmembrane domains of the ABCA transporter subfamily. The phylogenic tree of eukaryotic ABCA transporters based upon the NBD sequences, consists of three major clades, corresponding to the half-transporter single NBDs and to the full-transporter NBDls and NBD2s. A phylogenic tree of prokaryotic transporters and the eukaryotic ABCA transporters confirmed the evolutionary relationship between prokaryotic subfamily 7 transporters and eukaryotic ABCA half and full-transporters.

Interfacial exclusion pressure determines the ability of apolipoprotein A-IV truncation mutants to activate cholesterol ester transfer protein.

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.

Amoeboid Microglial Cells and not Astrocytes Synthesize TNF-alpha in Swiss Mouse Brain Cell Cultures.

The role of tumour necrosis factor (TNF-alpha) in brain physiology and pathology has been the focus of several studies. However, the source of this lymphokine in the central nervous system and the regulation of its synthesis is still poorly understood. We have therefore used purified astrocytes and brain macrophages in culture to compare the abilities of these two cell types to synthesize TNF-alpha and its mRNA. We find that, in the Swiss mouse, no significant TNF activity or TNF-alpha mRNA are produced by astrocytes, even following activation with lipopolysaccharides (LPS). On the other hand, purified microglial cells express a cytotoxic activity able to kill TNF-sensitive LM cells. Part of this activity is released into the culture medium and part remains bound to the membrane after mild paraformaldehyde treatment, demonstrating the existence in the culture of the soluble and membrane-bound forms of TNF activity. The fact that amoeboid microglial cells, and not astrocytes, are the actual source of TNF in brain cultures was further demonstrated by Northern blot analysis and in situ hybridization using a TNF-alpha specific oligonucleotide probe. The definition of the cell type which, in the CNS, is responsible for TNF synthesis will allow the regulation of this lymphokine to be analysed and opens the way for a better understanding of the interactions between amoeboid microglial cells and the other cell types which make up the nervous system.