Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis.

BACKGROUND: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis. METHODS: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. RESULTS: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). CONCLUSIONS: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID

A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat

Background: We aimed to further understand the relationship between cAMP concentration and mnesic performance. Methods and Findings: Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/ kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (,0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h)

Genome-wide mRNA expression profiling in vastus lateralis of COPD patients with low and normal fat free mass index and healthy controls

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) has significant systemic effects beyond the lungs amongst which muscle wasting is a prominent contributor to exercise limitation and an independent predictor of morbidity and mortality. The molecular mechanisms leading to skeletal muscle dysfunction/wasting are not fully understood and are likely to be multi-factorial. The need to develop therapeutic strategies aimed at improving skeletal muscle dysfunction/wasting requires a better understanding of the molecular mechanisms responsible for these abnormalities. Microarrays are powerful tools that allow the investigation of the expression of thousands of genes, virtually the whole genome, simultaneously. We aim at identifying genes and molecular pathways involved in skeletal muscle wasting in COPD. METHODS: We assessed and compared the vastus lateralis transcriptome of COPD patients with low fat free mass index (FFMI) as a surrogate of muscle mass (COPD(L)) (FEV(1) 30 ± 3.6%pred, FFMI 15 ± 0.2 Kg.m(−2)) with patients with COPD and normal FFMI (COPD(N)) (FEV(1) 44 ± 5.8%pred, FFMI 19 ± 0.5 Kg.m(−2)) and a group of age and sex matched healthy controls (C) (FEV(1) 95 ± 3.9%pred, FFMI 20 ± 0.8 Kg.m(−2)) using Agilent Human Whole Genome 4x44K microarrays. The altered expression of several of these genes was confirmed by real time TaqMan PCR. Protein levels of P21 were assessed by immunoblotting. RESULTS: A subset of 42 genes was differentially expressed in COPD(L) in comparison to both COPD(N) and C (PFP < 0.05; −1.5 ≥ FC ≥ 1.5). The altered expression of several of these genes was confirmed by real time TaqMan PCR and correlated with different functional and structural muscle parameters. Five of these genes (CDKN1A, GADD45A, PMP22, BEX2, CGREF1, CYR61), were associated with cell cycle arrest and growth regulation and had been previously identified in studies relating muscle wasting and ageing. Protein levels of CDKN1A, a recognized marker of premature ageing/cell cycle arrest, were also found to be increased in COPD(L). CONCLUSIONS: This study provides evidence of differentially expressed genes in peripheral muscle in COPD patients corresponding to relevant biological processes associated with skeletal muscle wasting and provides potential targets for future therapeutic interventions to prevent loss of muscle function and mass in COPD. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12931-014-0139-5) contains supplementary material, which is available to authorized users

As-Fabricated and Heat-Treated Microstructures of the Ti-6Al-4V Alloy Processed by Selective Laser Melting

International audienceSelective laser melting (SLM) is a rapid manufacturing process that enables the buildup of very complex parts in short delays directly from powder beds. Due to the high laser beam energy during very short interaction times and the high solidification rates of the melting pool, the resulting microstructure is out-of-equilibrium and particularly textured. This type of as-fabricated microstructure may not satisfy the aeronautical criterion and requires post heat treatments. Optimized heat treatments are developed, in one side, to homogenize and form the stable phases α and β while preventing exaggerated grain growth. In the other side, heat treatment is investigated to relieve the thermal stresses appearing during cooling. This study is aimed at presenting the various types of microstructure of the Ti-6Al-4V alloy after postfabrication heat treatments below or above the β transus. Tensile tests are then carried out at room temperature in order to assess the effect of the microstructures on the mechanical properties. The fine as-fabricated microstructure presents high yield and ultimate strengths, whereas the ductility is well below the standard. A strong anisotropy of fracture between the two loading directions is noted, which is attributed to the manufacturing defects. Conventional and optimized heat treatments exhibit high yield and ultimate strengths while the ductility is significantly improved. This is due to a new optimization of the process parameters allowing drastic reduction of the number of defects. These two heat treatments enable now a choice of the morphology of the grains between columnar or equiaxial as a function of the type of loading

Microstructural and mechanical approaches of the selective laser melting process applied to a nickel-base superalloy

International audienceThis article aims at presenting the Nimonic 263 as-processed microstructure of the selective laser melting which is an innovative process. Because the melting pool is small and the scanning speed of the laser beam is relatively high, the as-processed microstructure is out-of-equilibrium and very typical to additive manufacturing processes. To match the industrial requirement, the microstructures are modified through heat treatments in order to either produce precipitation hardening or relieve the thermal stresses. Tensile tests at room temperature give rise to high mechanical properties close or above those presented by Wang et al. [1]. However, it is noted a strong anisotropy as a function of the building direction of the samples because of the columnar grain growth

The prospects for additive manufacturing of bulk TiAl alloy

International audienceThis paper deals with the prospects for additive manufacturing (AM) of bulk TiAl alloys. A number of AM processes have already been explored in the literature for these intermetallic alloys. The main trend developed in published works concerns a strong crack sensitivity of this relatively brittle material due to rapid successive heating and cooling cycles. Optimized processing conditions have already been achieved for producing sound and crack-free TiAl materials by means of EBM and LMD processes. This experimental work was particularly focused on the third process, i.e. SLM, to produce fully dense TiAl parts. A series of beads, surface layers and cubes have been manufactured to investigate the microstructural evolution. Post heat treatments allowed a uniform microstructure to be restored for the intermetallic TiAl alloy

Direct fabrication of a Ti-47Al-2Cr-2Nb ally by selective laser melting and direct metal deposition processes

International audienceA Ti-47Al-2Cr-2Nb (at.%) material was fabricated using two laser-based methods, “Selective Laser Melting” (SLM) and “Direct Metal Deposition” (DMD), for potential uses in aircraft jet engines. Experiments were conducted under controlled atmosphere by changing the processing parameters. Optimal parameters were searched for this relatively low ductility material to prevent cracking due to built-up residual stresses during fast cooling. It was observed that these non-equilibrium cooling conditions were fast enough to generate ultra fine and metastable structures exhibiting high microhardness values. Post heat-treatments were successfully used to restore homogeneous lamellar or duplex microstructures and to relieve the residual stresses. A comparison of these two methods is provided in terms of powder requirements and of process parameters to achieve noncracked structures and fully dense materials