Single, short in-del, and copy number variations detection in monogenic dyslipidemia using a next-generation sequencing strategy.

Optimal molecular diagnosis of primary dyslipidemia is challenging to confirm the diagnosis, test and identify at risk relatives. The aim of this study was to test the application of a single targeted next-generation sequencing (NGS) panel for hypercholesterolemia, hypocholesterolemia, and hypertriglyceridemia molecular diagnosis. NGS workflow based on a custom AmpliSeq panel was designed for sequencing the most prevalent dyslipidemia-causing genes (ANGPTL3, APOA5, APOC2, APOB, GPIHBP1, LDLR, LMF1, LPL, PCSK9) on the Ion PGM Sequencer. One hundred and forty patients without molecular diagnosis were studied. In silico analyses were performed using the NextGENe software and homemade tools for detection of copy number variations (CNV). All mutations were confirmed using appropriate tools. Eighty seven variations and 4 CNV were identified, allowing a molecular diagnosis for 40/116 hypercholesterolemic patients, 5/13 hypocholesterolemic patients, and 2/11, hypertriglyceridemic patients respectively. This workflow allowed the detection of CNV contrary to our previous strategy. Some variations were found in previously unexplored regions providing an added value for genotype-phenotype correlation and familial screening. In conclusion, this new NGS process is an effective mutation detection method and allows better understanding of phenotype. Consequently this assay meets the medical need for individualized diagnosis of dyslipidemia.

Assessment of changes in lactate concentration with intravascular microdialysis during high-risk cardiac surgery using the trend interchangeability method.

Background: Blood lactate is a strong predictor of mortality, and repeated blood lactate assays are recommended during surgery in high-risk patients. We hypothesized that the use of intravascular microdialysis incorporated in a central venous catheter would be interchangeable with the reference blood gas technique to monitor changes in blood lactate. Methods: Microdialysis and central venous blood lactate measurements were recorded simultaneously in high-risk cardiac surgical patients. The correlation between absolute values was determined by linear regression, and the Bland-Altman test for repeated measurements was used to compare bias, precision, and limits of agreement. Changes in lactate measurements were evaluated with a four-quadrant plot and trend interchangeability method (TIM). Results: In the 23 patients analysed, the central venous catheter was used as part of standard care, with no complications. The correlation coefficient for absolute values ( n =104) was 0.96 ( P <0.0001). The bias, precision, and limits of agreement were -0.19, 0.51, and -1.20 to 0.82 mmol litre -1 , respectively. The concordance rate for changes in blood lactate measurements ( n =80) was 94% with the four-quadrant plot. In contrast, the TIM showed that 23 (29) changes in lactate measurements were not interpretable, and among the remaining 57 (71) interpretable changes, 18 (32) were interchangeable, 8 (14) were in the grey zone, and 31 (54) were not interchangeable. Conclusions: Microdialysis with a central venous catheter appears to provide reliable absolute blood lactate values. Although changes in blood lactate measurements showed an excellent concordance rate, changes between the two methods were poorly interchangeable with the TIM. Clinical trial registration: NCT02296593.

Influence of bacterial resistance on mortality in intensive care units: a registry study from 2000 to 2013 (IICU Study).

BACKGROUND: Bacterial resistance to antibiotics is a daily concern in intensive care units. However, few data are available concerning the clinical consequences of in-vitro-defined resistance. AIM: To compare the mortality of patients with nosocomial infections according to bacterial resistance profiles. METHODS: The prospective surveillance registry in 29 French intensive care units (ICUs) participating during the years 2000-2013 was retrospectively analysed. All patients presenting with a nosocomial infection in ICU were included. FINDINGS: The registry contained 88,000 eligible patients, including 10,001 patients with a nosocomial infection. Among them, 3092 (36.7%) were related to resistant micro-organisms. Gram-negative bacilli exhibited the highest rate of resistance compared to Gram-positive cocci (52.8% vs 48.1%; P < 0.001). In-hospital mortality was higher in cases of patients with antibiotic-resistant infectious agents (51.9% vs 45.5%; P < 0.001), and critical care length of stay was longer (33 ± 26 vs 29 ± 22 days; P < 0.001). These results remained significant after SAPS II matching (P < 0.001) and in the Gram-negative bacilli and Gram-positive cocci subgroups. No difference in mortality was found with respect to origin prior to admission. CONCLUSION: Patients with bacterial resistance had higher ICU mortality and increased length of stay, regardless of the bacterial species or origin of the patient.

Physico-chemical studies of resveratrol, methyl-jasmonate and cyclodextrin interactions: an approach to resveratrol bioproduction optimization.

trans-Resveratrol (RSV) is a natural phenolic molecule of the stilbene family known for its anti-oxidant properties in the field of nutraceuticals and cosmetics. Its production by grapevine cell suspensions is induced by the addition to the culture medium of elicitor compounds, methyl jasmonate (MeJA) and cyclodextrins (CDs). Physico-chemical studies were performed to understand the mechanism of action of CDs on this bioproduction of RSV. Inclusion complexes of RSV in CDs were first observed and then interactions with MeJA were identified using various analytical techniques such as UV and nuclear magnetic resonance (NMR) spectroscopies, mass spectrometry (MS) and isothermal titration calorimetry (ITC).

[Biochemical and molecular diagnosis of Gaucher disease in Tunisia]. / Diagnostic biochimique et moléculaire de la maladie de Gaucher en Tunisie.

Our study was carried out at a family from the Sahel (Tunisia). The father (index case) and his two children (son and daughter). The father beta-glucocerebrosidase (GCB) activity showing a deficit. These biochemical analyses are supplemented by molecular studies: enzymatic digestion and the direct sequencing. Two mutations were analysed, the p.Asn 370 Ser and the p.Leu 444 Pro. The DNA sequencing confirmed the presence of the homozygous genotype of this p.Asn 370 Ser in the father DNA and the heterozygous one in the two children DNA. It has no detection of the 55 pb deletion in exon 9 among all the specimens of DNA treated. The mutation p.Asn 370 Ser is associated with Gaucher disease type 1 correlated of a total absence of neurological involvements.

[Mucopolysaccharidosis type I: identification of alpha-L-iduronidase mutations in Tunisian families]. / La mucopolysaccharidose de type I: identification des mutations du gène alpha-L-iduronidase dans des familles tunisiennes.

UNLABELLED: Mucopolysaccharidosis type I (MPS I) is a lysosomal disease due to mutations in the gene encoding alpha-l-iduronidase (IDUA) leading to variable clinical phenotypes with progressive severe organomegaly, bone and neurological involvement in the most severe forms. The aim of our study was to propose in Tunisia a strategy of molecular and prenatal diagnosis of the MPS I. POPULATION AND METHODS: Our study was carried out on 8 MPS I patients recruited from different Tunisian regions and issued from 5 unrelated families. All the patients were offspring of consanguineous marriages. RESULTS: The clinical and biological study led to diagnose 5 Hurler patients and 3 Hurler-Scheie patients. Three IDUA mutations were identified by molecular analysis within 6 different families: a novel mutation p.F602X and 2 already described mutations p.P533R and p.R628X. DISCUSSION: MPS I is a heterogeneous disease characterized by variability of the phenotypes. The missense mutation p.P533R associated with the intermediate phenotype was the most frequent in the Tunisian but also in the Moroccan population. In Tunisia, the incidence of p.P533R mutation seems to be associated with the high frequency of consanguineous marriages. CONCLUSION: The identification of known MPS I mutations (p.P533R and p.R628X) and of the novel mutation p.F602X permits reliable genetic counselling of at-risk relatives and molecular prenatal diagnosis.

A constrained extended Kalman filter for the optimal estimate of kinematics and kinetics of a sagittal symmetric exercise.

This paper presents a method for real-time estimation of the kinematics and kinetics of a human body performing a sagittal symmetric motor task, which would minimize the impact of the stereophotogrammetric soft tissue artefacts (STA). The method is based on a bi-dimensional mechanical model of the locomotor apparatus the state variables of which (joint angles, velocities and accelerations, and the segments lengths and inertial parameters) are estimated by a constrained extended Kalman filter (CEKF) that fuses input information made of both stereophotogrammetric and dynamometric measurement data. Filter gains are made to saturate in order to obtain plausible state variables and the measurement covariance matrix of the filter accounts for the expected STA maximal amplitudes. We hypothesised that the ensemble of constraints and input redundant information would allow the method to attenuate the STA propagation to the end results. The method was evaluated in ten human subjects performing a squat exercise. The CEKF estimated and measured skin marker trajectories exhibited a RMS difference lower than 4mm, thus in the range of STAs. The RMS differences between the measured ground reaction force and moment and those estimated using the proposed method (9N and 10Nm) were much lower than obtained using a classical inverse dynamics approach (22N and 30Nm). From the latter results it may be inferred that the presented method allows for a significant improvement of the accuracy with which kinematic variables and relevant time derivatives, model parameters and, therefore, intersegmental moments are estimated.

Clinical, morphological, and molecular aspects of sialic acid storage disease manifesting in utero.

BACKGROUND: Sialic acid storage diseases (SASDs) are caused by the defective transport of free sialic acid outside the lysosome. Apart from the Salla presentation in Finland, SASD is a very rare form of lysosomal storage disease (LSD) with approximately 35 cases, all diagnosed after birth, having been reported worldwide. We report a series of 12 French patients with very early manifestations, including eight fetuses diagnosed in utero. RESULTS: Ultrasound examination, fetal autopsy, or clinical examination showed prominent ascites, rarely progressing to complete hydrops, and highlighted the early severity of bone disease. Dramatic increase of free sialic acid in various biological samples confirmed the diagnosis in all cases. Storage staining affinities and storage distribution in placenta and fetal organs allowed differential diagnosis from other LSDs but cannot differentiate between SASD, sialidosis, and galactosialidosis. Fourteen different mutations were identified, showing the molecular heterogeneity of SASD in the French population. We found that the previously described p.Y306X mutation generated two different transcripts, and we identified seven novel mutations: three deletions (del exon 7, del exons10+11 and c.1296delT), one splice site mutation (c.1350+1G-->T) one nonsense mutation (p.W339X), and two missense mutations (p.R57C and p.G127E). CONCLUSIONS: The severity of our patients' genotypes is in agreement with their phenotypes but not with the importance and early appearance of the very frequent in utero manifestations. Minimal fetal disease in some patients and a reported case of heterogeneity of fetal involvement within a family suggest that factors other than the genotype influence fetal manifestations.

A new 3D center of mass control approach for FES-assisted standing: First experimental evaluation with a humanoid robot.

This paper proposes a new control framework to restore the coordination between upper (functional) and lower (paralyzed) limbs in the context of functional electrical stimulation in completely paraplegic individuals. A kinematic decoupling between the lower and upper limbs controls the 3D whole-body center of mass location and the relative foot positions by acting only on the lower-limb joints. The upper limbs are free to move under voluntary control, and are seen as a perturbation for the lower limbs. An experimental validation of this paradigm using a humanoid robot demonstrates the real-time applicability and robustness of the method. Different scenarios mimicking the motion of a healthy subject are investigated. The proposed method can maintain bipedal balance and track the desired center of mass trajectories under movement disturbances of the upper limbs with an error inferior to 0.01 m under any conditions.

COS cell expression studies of P86L, P86R, P480L and P480Q Hunter's disease-causing mutations.

Three missense mutations identified in the IDS gene of our Hunter's disease patients (P86L, P480L and P480Q) and the previously described P86R mutation were expressed in COS cells to evaluate their functional consequence on iduronate-2-sulfatase (IDS) activity and processing. The 86-proline residue belongs to the highly conserved pentapeptide C-X-P-S-R in which cysteine modification to a formylglycine is required for sulfatase activity. The substitution of the 86-proline residue led to a severe mutation as no mature form was targeted to the lysosome in agreement with the severe phenotype observed in patients carrying P86L and P86R mutations. Expression studies with P480L and P480Q mutant cDNAs showed the presence of a small amount of 55 kDa mature form in the lysosomes of transfected COS cells. IDS activity of the P480L and P480Q mutants in cell extracts represents 16.6% and 5.4% of the wild-type, respectively.

Hypogravity increases cyclopiazonic acid sensitivity of rat soleus muscle.

The functional capacity of skeletal muscle sarcoplasmic reticulum was explored in slow rat soleus muscle after 21 days of hindlimb suspension. The sarcoplasmic reticulum function was assessed in intact and saponin-skinned fibers by using cyclopiazonic acid, a specific Ca(2+)-adenosinetriphosphatase inhibitor. After hindlimb unweighting, the sensitivity to cyclopiazonic acid of intact and skinned soleus fibers becomes similar to that found in fast-twitch muscles. This change could be related to the expression of fast Ca2(+)-adenosinetriphosphatase-pump protein in unloaded soleus muscles and agrees with a transformation of soleus muscle from slow- to fast-twitch type. These results also indicate that specific pharmacological tools, like cyclopiazonic acid, could be used to analyze subcellular functional changes due to hindlimb unweighting.

Potentiation of the twitch responses by inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase in frog atrial fibres.

In frog atrial fibres, cyclopiazonic acid as well as thapsigargin, which are inhibitors of sarcoplasmic reticulum Ca(2+)-ATPase, induced a significant increase in the twitch amplitude without detectable changes in its kinetics. The measurements performed on chemically skinned fibres show that cyclopiazonic acid has no effect on the properties of contractile proteins. In the presence of a T-type Ca2+ channel blocker or L-type Ca2+ channel blocker, cyclopiazonic acid still induced a potentiation of the twitch while no effect was found in the presence of a Na(+)-Ca2+ exchange blocker. The effect of cyclopiazonic acid was not related to any modification in myofibrillar Ca2+ sensitivity or in Ca2+ influx through Ca2+ channels. It is proposed that the inhibition of the sarcoplasmic reticulum Ca(2+)-ATPase resulted in a potentiation of the effect of the Ca2+ influx and that the major role of the sarcoplasmic reticulum was to limit the intracellular Ca2+ concentration.

Increased synovial fluid levels of interleukin-12, sCD25 and sTNF-RII/sTNF-RI ratio delineate a cytokine pattern characteristic of immune arthropathies.

The assessment of cytokines and their soluble receptors in the synovial fluid (SF) of inflammatory arthropathies may be useful in studying pathogenetic and immunoregulatory mechanisms underlying different diseases. The aim of this work was to study the cytokine network occurring in inflammatory arthropathies and to identify a cytokine profile which is characteristic of an immune-mediated synovitis. Levels of IL-12, as well as IL-4, IL-8, IL-10, IFN-gamma, sCD25, TNF-alpha and its soluble receptors were measured in the SF of various arthropathies, i.e. non-inflammatory arthropathies: "control" meniscus pathology (n = 21), osteoarthritis (n = 22) and chronic crystal arthritis (n = 9); a non-immune inflammatory arthropathy: acute crystal arthritis (n = 11); 2 immune inflammatory arthropathies: reactive arthritis (ReA) (n = 23) and rheumatoid arthritis (RA) (n = 44). SF levels of IL-10, TNF-alpha and sTNF-RII were found to be increased in the three inflammatory arthropathies compared to the "control" meniscus group. Within the inflammatory group, acute crystal arthritis was characterized by a significantly higher sTNF-RI/TNF-alpha ratio and ReA by a significantly lower sTNF-RII/TNF-alpha ratio compared to the two other diseases. The two immune arthropathies, RA and ReA, were characterized by increased SF levels of IL-12, sCD25 and of the sTNF-RII/sTNF-RI ratio. ReA differed however from RA by showing lower IL-8 and IL-4 levels, higher IFN-gamma levels and a higher IL-12/IL-10 ratio, suggesting a more prevalent Th1 profile in ReA SF. Our data indicate that the measurement of SF cytokines and soluble receptors may discriminate between each inflammatory arthropathy and might be useful in clinical practice.

Seabirds drive plant species turnover on small Mediterranean islands at the expense of native taxa.

The analysis of long-term floristic changes was conducted on nine west-Mediterranean limestone islands (size range: 2-95 ha) which have recently undergone a severe demographic explosion in their yellow-legged gull Larus cachinnans colonies. A comparison of past and present plant inventories was used to quantify extinction-colonization events, both from a classical biogeographical perspective (per island approach) and a metapopulational perspective (per species approach). In the first approach, floristic turnover intensity was negatively related to island area and positively to gull nesting density, but was independent of island isolation. In the second, species turnover rate was compared with a set of plant species life history traits (dispersal mode, Grime CSR strategy, growth form, biogeographical type). Plants which exhibited the highest turnover rate were primarily ruderal, annual, wind-dispersed species with a wide geographic range. The severe disturbance induced by seabird activities has tended to select and favour some adapted plant species groups at the expense of indigenous island taxa. The relationships between specific turnover intensity and plant life history traits justify using the metapopulation approach and point to the importance of interspecific variations in extinction-colonization patterns.

[Evaluating radiotherapy patients' need for information: a study using a patient information booklet] . / Evaluation des besoins en information des patients suivis en radiothérapie: étude effectuée sur la base du livret de la radiothérapie.

The French Radiotherapy-Oncology Society (SFRO) and the National Trade of Radiotherapists-Oncologists (SNRO) elaborated and published a patient information booklet on radiotherapy, in 1999. This present study appraises the pertinence of the form and substance of this booklet one year after its release. Eight radiotherapy centers participated in this research which evaluated 162 patients at treatment initiation. The conclusions of this study demonstrated the importance of clearly informing patients of their disease, treatment, and the secondary effects of treatment. It is essential to emphasize that 97% of the patients declared that an information booklet is a real necessity, and that the one provided by the SFRO responds to the majority of their concerns. Obtaining technical and practical knowledge resulted in a reassurance about their treatment. The most revealing result is that 87% requested direct communication about their illness, and that cancer be named by this word and not other, evasive terms. Seventy-two percent of the patients requested more information about their cancer, different treatment options, and quality of life issues in an attempt to psychologically prepare themselves to face an illness for which they have little control. Patients refuse to be passive, and claim the right to become 'partners' of the medical teams, concerning their treatment and recovery.

[Lipid balance in an arteritic patient and therapeutic implications]. / Quel bilan lipidique chez un artéritique et quelles implications thérapeutiques.

The pathogenesis of atherosclerosis remains hypothetical, the current vogue of a lipidic origin being now only a working hypothesis. The management of an arteritic patient (for a treating clinician) is that of a case of global atherosclerosis. Screening for lipids: 1) is here of only moderate interest in the close scrutiny of currently recognized risk factors, and 2) should include initially a simple study of total plasma cholesterol (TC) and triglycerides (TG) on two occasions at one month's interval in a reliable laboratory. Therapeutic implications are a function of a global analysis of the patient and not just the values for even sophisticated assays of circulating lipids.

[Obstetric analgesia in patients with Klippel-Trenaunay syndrome]. / Analgésie obstétricale chez des patientes atteintes d'un syndrome de Klippel-Trenaunay.

We report three cases of delivery in two parturients with a Klippel-Trenaunay syndrome. These patients have a rare hereditary disorder that results in three main features: haemangiomas, varicose veins, bone and soft tissue hypertrophy. In the absence of angiographic magnetic resonance imaging of the spinal cord and of perispinal tissues, arteriovenous malformations of the central nervous system could not been ruled out. Intravenous sufentanil and pudendal block were used for labour analgesia and vaginal delivery respectively; general anaesthesia was used for uterine revision and for caesarean section.

[Learning placement of a new oropharyngeal intubation (Copa)]. / Apprentissage de la mise en place d'une nouvelle canule oropharyngée (Copa).

OBJECTIVE: To assess learning of Copa (cuffed oropharyngeal airway) technique required for insertion and manual ventilation. STUDY DESIGN: Open prospective clinical study. PERSONS: Four staff members, two anaesthetists and two nurse anaesthetists and 74 ASA I or II patients without difficult intubation criteria presenting for minor surgery in the supine position under general anaesthesia. METHODS: After induction of anaesthesia, the Copa was inserted by each doctor and nurse in at least 16 patients and the ventilation manually assisted until spontaneous breathing resumed. RESULTS: The success rate of insertion at the first attempt was 97%. Therefore no learning period was required. Two laryngospasms and two episodes of cough occurred during Copa placement. Additional manipulations to optimise the ventilation were needed in 48 patients. CONCLUSION: Copa is easy to use without previous training. Further studies are required to assess its side effects and the appropriate depth of anaesthesia required for its insertion.

Assessing neuromuscular mechanisms in human-exoskeleton interaction.

In this study, we propose to evaluate a 7 DOF exoskeleton in terms of motion control. Using criteria from the human motor control literature, inverse optimization was performed to assess an industrial screwing movement. The results of our study show that the hybrid composition of the free arm movement was accurately determined. At contrary, when wearing the exoskeleton, which produces an arbitrary determined torque compensation, the motion is different from the naturally adopted one. This study is part of the evaluation and comprehension of the complex neuromuscular mechanism resulting in wearing an exoskeleton several hours per day for industrial tasks assistance.

Toward an affordable and user-friendly visual motion capture system.

The present study aims at designing and evaluating a low-cost, simple and portable system for arm joint angle estimation during grasping-like motions. The system is based on a single RGB-D camera and three customized markers. The automatically detected and tracked marker positions were used as inputs to an offline inverse kinematic process based on bio-mechanical constraints to reduce noise effect and handle marker occlusion. The method was validated on 4 subjects with different motions. The joint angles were estimated both with the proposed low-cost system and, a stereophotogrammetric system. Comparative analysis shows good accuracy with high correlation coefficient (r= 0.92) and low average RMS error (3.8 deg).