VECMAtk: a scalable verification, validation and uncertainty quantification toolkit for scientific simulations.

We present the VECMA toolkit (VECMAtk), a flexible software environment for single and multiscale simulations that introduces directly applicable and reusable procedures for verification, validation (V&V), sensitivity analysis (SA) and uncertainty quantication (UQ). It enables users to verify key aspects of their applications, systematically compare and validate the simulation outputs against observational or benchmark data, and run simulations conveniently on any platform from the desktop to current multi-petascale computers. In this sequel to our paper on VECMAtk which we presented last year [1] we focus on a range of functional and performance improvements that we have introduced, cover newly introduced components, and applications examples from seven different domains such as conflict modelling and environmental sciences. We also present several implemented patterns for UQ/SA and V&V, and guide the reader through one example concerning COVID-19 modelling in detail. This article is part of the theme issue 'Reliability and reproducibility in computational science: implementing verification, validation and uncertainty quantification in silico'.

Hereditary angioedema with F12 mutation: factors modifying the clinical phenotype.

BACKGROUND: Hereditary angioedema (HAE) with normal C1 inhibitor (C1Inh) associated with the c.983C>A and c.983C>G mutations of the F12 gene (FXII-HAE) is a rare condition, and presents with highly variable clinical expression. On the basis of data gathered from a large carrier cohort, we assessed the modifiers affecting the clinical phenotype. METHODS: We analyzed clinical and biological data recorded from 118 mutation carriers (80 symptomatic and 38 asymptomatic), 58 noncarrier relatives from 40 families, and 200 healthy donors. Disease severity was scored in relation to frequency and location of edema, as well as age at disease onset. To predict FXII-HAE disease severity, we analyzed the biological phenotype [C1Inh, C4, spontaneous amidase, angiotensin-I-converting enzyme (ACE), aminopeptidase P (APP), and carboxypeptidase N/M (CPN)] by means of logistic regression (Akaike information criterion) and odds ratio (OR). RESULTS: Meaningful variables contributed to FXII-HAE, with the kinin catabolism enzymes ACE and CPN exhibiting a significant inverse relationship with disease severity (OR = 0.36, 95% CI 0.23-0.59, P < 0.001; OR = 0.58, 95% CI 0.36-0.91, P < 0.05, respectively). CPN activities were 37.5 (28.5-41.3) nmol/ml/min and 38.5 (32.8-45.6) for FXII-HAE asymptomatic and symptomatic carriers, respectively, and 37.9 (30.5-43.7) nmol/ml/min for noncarriers. Angiotensin-I-converting enzyme activities were 58 (44-76) and 49 (35-59) nmol/ml/min for FXII-HAE asymptomatic and symptomatic carriers, respectively, and 56 (49-66) nmol/ml/min for noncarriers. CONCLUSIONS: The FXII-HAE is associated with modifiers, for example kinin catabolism enzymes, ACE and CPN, different from those recognized in HAE with C1Inh deficiency.

Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization.

AIMS: To report the clinical, pathological and genetic findings in a group of patients with a previously not described phenotype of congenital myopathy due to recessive mutations in the gene encoding the type 1 muscle ryanodine receptor channel (RYR1). METHODS: Seven unrelated patients shared a predominant axial and proximal weakness of varying severity, with onset during the neonatal period, associated with bilateral ptosis and ophthalmoparesis, and unusual muscle biopsy features at light and electron microscopic levels. RESULTS: Muscle biopsy histochemistry revealed a peculiar morphological pattern characterized by numerous internalized myonuclei in up to 51% of fibres and large areas of myofibrillar disorganization with undefined borders. Ultrastructurally, such areas frequently occupied the whole myofibre cross section and extended to a moderate number of sarcomeres in length. Molecular genetic investigations identified recessive mutations in the ryanodine receptor (RYR1) gene in six compound heterozygous patients and one homozygous patient. Nine mutations are novel and four have already been reported either as pathogenic recessive mutations or as changes affecting a residue associated with dominant malignant hyperthermia susceptibility. Only two mutations were located in the C-terminal transmembrane domain whereas the others were distributed throughout the cytoplasmic region of RyR1. CONCLUSION: Our data enlarge the spectrum of RYR1 mutations and highlight their clinical and morphological heterogeneity. A congenital myopathy featuring ptosis and external ophthalmoplegia, concomitant with the novel histopathological phenotype showing fibres with large, poorly delimited areas of myofibrillar disorganization and internal nuclei, is highly suggestive of an RYR1-related congenital myopathy.

Mutational spectrum and phenotypes in Danish families with hereditary angioedema because of C1 inhibitor deficiency.

BACKGROUND: Hereditary angioedema (HAE), type I and II, is an autosomal dominant disease with deficiency of functional C1 inhibitor protein causing episodic swellings of skin, mucosa and viscera. HAE is a genetically heterogeneous disease with more than 200 different mutations in the SERPING1 gene. A genotype-phenotype relationship does not seem to exist in HAE, although the polymorphism c.-21T>C of exon 2 has been reported to be associated with a more severe phenotype. We aimed to establish the mutational spectrum of C1 inhibitor deficiency in Denmark and investigate the possible disease-aggravating effect of the c.-21T>C polymorphism. METHODS: Hereditary angioedema was diagnosed based on clinical features and C1 inhibitor deficiency. A general severity score ranging from 0 to 10 was developed based on age at disease onset, clinical manifestations and treatment experiences. SERPING1 gene investigation was performed by exon sequencing followed by multiplex ligation-dependent probe amplification genomic rearrangement analysis in all known Danish HAE families. RESULTS: Fifty-nine patients with HAE from 26 families were included in this study. The mean disease severity score was 7.12 [1-10], and the mean C1 inhibitor function was 26% [20-46%]. The sensitivity of the mutational screening was 96%, and 13 new mutations were found in this Danish patient cohort. Nine patients (15%) carried the c.-21T>C polymorphism, but they didn't have a more severe phenotype. CONCLUSION: Thirteen new mutations were identified in the Danish HAE population. No correlation between the c.-21T>C polymorphism, the biochemical values of C1 inhibitor function and the clinical severity score was found.

Early onset of hypokalaemic periodic paralysis caused by a novel mutation of the CACNA1S gene.

We report a precocious and atypical form of hypokalaemic periodic paralysis, with clinical manifestations at birth and first episodes of paralysis occurring as early as 1 year of age, although onset of this disease usually occurs between 5-35 years. Extensive molecular analysis showed that the disease was caused by a novel de novo p.Arg897Ser mutation in the CACNA1S gene. The mutation mapped to a new region of the protein, the S4 voltage sensing segment of domain III, at odds with previously reported mutations that exclusively affected domains II and IV.

[Oculo-cerebro-renal Lowe syndrome: clinical, biochemical and molecular studies in a Moroccan patient]. / Syndrome oculo-cérébro-rénal de Lowe: étude clinique, biochimique et moléculaire chez un patient marocain.

The oculo-cerebro-renal syndrome of Lowe is a rare X-linked disorder, caused by the inositol biphosphate 5-phosphatase deficiency, localized to the Golgi complex. Several mutations were reported in patient's OCRL gene leading to enzyme deficiency. We report a Moroccan case of OCRL syndrome of Lowe with a neo mutation in exon 10. The patient aged of 19 months was referred to our medical centre because of a psychomotor retardation. He had a medical history of eye abnormalities including cataract and bilateral glaucoma, diagnosed when he was 5 weeks old. Cataract has been treated after chirurgical therapy but ocular hypertonia persisted. Physical examination revealed an axial hypotonia and walking difficulties. Laboratory tests revealed a moderate acidosis (20 mmol/L), a slight decrease of serum phosphate level (24 mg/L) and an increased serum phosphatase activity. Further studies showed mild proteinuria, urinary bicarbonates loosing and generalised hyperaminoaciduria. Based on both clinical and biological data, Lowe syndrome has been suggested. In this context, molecular investigation has been performed using dHPLC/sequencing techniques which allow identifying an original mutation c.776T>C (p.Phe259Ser), localized on the exon 10 of the OCRL gene. The mutation was not found in the probant's mother suggesting a neo mutation. Lowe syndrome is a rare hereditary X-linked disorder resulting from a variety of heterogeneous mutations of OCRL gene. Indeed, numerous mutations have been reported, variations were noted concerning their localization as well as their type. To our knowledge, this is the first report of the neo mutation c.776T>C of OCRL gene and the first published case report of the Lowe syndrome in a Moroccan patient.

Electron spin resonance study of human transcortin: Thiol groups and binding site topography.

A series of cortisol analogs bearing a nitroxide free radical on C-17 side chains with a variation of distance between the steroid D-ring and the spin label from 7.4 to 17.6 A has been synthesized. These analogs were found to retain a good affinity for the specific corticosteroid binding site of purified human transcortin. The spin-labeled cortisol analogs were used to probe the human transcortin binding site structure by electron spin resonance (ESR) spectroscopy. A total depth of approx. 25 A was estimated for the binding site crevice. Use of sulfhydryl reagents (N-ethylmaleimide, p-chloromercuribenzoate) showed that a maximum of two sulfhydryl groups were titratable after reduction and denaturation of the protein. One of these thiol groups appeared to be involved in the cortisol binding site and could not be detected in the presence of bound steroid. ESR study of its environment, using spin-labeled N-ethylmaleimide reagents of various side-chain lengths, led to the conclusion that this thiol was at a depth of approx. 15 A or more in the binding site cavity. The second sulfhydryl group may be present in an oxidized form in the purified native transcortin, since it became titratable only after reductive treatment of the protein. ESR study showed that this thiol may be located in a crevice at approx. 15 A from the protein surface. These findings are compatible with a structural organization of the transcortin cortisol binding site, taking into account tentative models previously proposed by others.

Molecular organization (topography) of cytochrome P-450(11)beta in mitochondrial membrane and phospholipid vesicles as studied by trypsinolysis.

Cytochrome P-450(11)beta from adrenal cortex is an intrinsic membrane protein embedded in the inner mitochondrial membrane. Topography of the protein inside a phospholipid bilayer was examined using controlled proteolysis of purified cytochrome P-450(11)beta following its integration into artificial liposomes. Inclusion of the protein into phospholipid vesicles led to a marked stabilization of the cytochrome activity. Trypsin treatment of the liposome-integrated cytochrome resulted in the rapid disappearance of the native protein moiety (47 kDa), while a major 34 kDa peptide component was formed. This peptide core retained the heme moiety and part of the cytochrome steroid-11 beta hydroxylase activity. Very similar observations were obtained when inside-out vesicles prepared from isolated adrenocortical mitoplasts were examined with the same approach. It is thus suggested that adrenocortical cytochrome P-450(11)beta is embedded in the inner mitochondrial membrane as well as in artificial liposomes by a major hydrophobic domain associated with the heme moiety while a limited domain remains accessible on the matrix side of the membrane surface. The previous described phosphorylation of the cytochrome P-450(11)beta on a serine residue, by the cAMP-dependent protein kinase is suggested to occur in the protein domain oriented toward the membrane surface, the phosphorylation site being lost under mild proteolytic digestion of the membrane-integrated protein.

Clinical and histopathological aspects of central core disease associated and non-associated with RYR1 locus.

We analysed the clinical, histochemical, ultrastructural and genetic data of patients affected by central core disease (CCD) studied during the last 20 years. From a total series of 86 CCD-families, we have identified 46 CCD families with RYR1 mutations (16 autosomal dominant, 8 autosomal recessive, 17 sporadic cases and 5 de novo mutations). Out of the other 40 CCD families, the RyR1 gene was entirely excluded in 7 families, by cDNA sequencing or linkage analysis, indicating a genetic heterogeneity of CCD.

Evaluation and clinical interest of mannan binding lectin function in human plasma.

The mannan binding lectin (MBL) plays a major role in innate immunity through its ability to activate complement upon binding to carbohydrate arrays on the surface of various microorganisms. The question of a possible association of the MBL structural gene polymorphism and the oligomeric state of MBL was poorly documented. For these reasons, it appears difficult to evaluate MBL in blood patients on the only basis of protein contents, even in combination with MBL genotyping. This study reports a method to calculate a specific activity for circulating MBL, that relies on: (i) the availability of purified MBL; and (ii) a simplified MBL activity assay based on complement activation. The three-step MBL purification from human plasma reported here is characterized by a highly purified MBL, that occurs in two different oligomeric forms. The results on the specific activity of these forms show that the higher oligomeric forms of MBL have the ability to induce C4 cleavage more efficiently than the corresponding lower oligomers. The usefulness of this approach is illustrated by its potential interest in the biological exploration of certain pathology, for example in the follow-up of chronic hepatitis C. Further investigation is needed to establish whether MBL specific activity (MBLsa) is correlated to the polymorphic state of the molecule. The relative simplicity of the test described here allows better investigation on the relationship between MBL biological activity and its genotype.

Recent advances in the diagnosis of malignant hyperthermia susceptibility: how confident can we be of genetic testing?

Malignant hyperthermia (MH) is a condition that manifests in susceptible individuals only on exposure to certain anaesthetic agents. Although genetically heterogeneous, mutations in the RYR1 gene (19q13.1) are associated with the majority of reported MH cases. Guidelines for the genetic diagnosis for MH susceptibility have recently been introduced by the European MH Group (EMHG). These are designed to supplement the muscle biopsy testing procedure, the in vitro contracture test (IVCT), which has been the only means of patient screening for the last 30 years and which remains the method for definitive diagnosis in suspected probands. Discordance observed in some families between IVCT phenotype and susceptibility locus genotype could limit the confidence in genetic diagnosis. We have therefore assessed the prevalence of 15 RYR1 mutations currently used in the genetic diagnosis of MH in a sample of over 500 unrelated European MH susceptible individuals and have recorded the frequency of RYR1 genotype/IVCT phenotype discordance. RYR1 mutations were detected in up to approximately 30% of families investigated. Phenotype/genotype discordance in a single individual was observed in 10 out of 196 mutation-positive families. In five families a mutation-positive/IVCT-negative individual was observed, and in the other five families a mutation-negative/IVCT-positive individual was observed. These data represent the most comprehensive assessment of RYR1 mutation prevalence and genotype/phenotype correlation analysis and highlight the possible limitations of MH screening methods. The implications for genetic diagnosis are discussed.

[Transaminase B from Escherichia coli. I.-Purification and first properties]. / Transaminase B d'Escherichia coli. I. - Purification et premières propriétés

Transaminase B (EC.2.6.1.6.) from E. coli, the specific enzyme for branched-chain aminoacids, was obtained in a purity equal to or greater than 96 p. cent after an 800-fold purification, employing two different procedures. One of the procedures involved heating at 60degreesC. The apparent molecular weight of the enzyme was estimated by chromatography on Sephadex and gel electrophoresis to be close to 180,000. The protein is made up of 6 subunits of equal size, with one molecule of coenzyme in each. Its absorption spectrum shows bands at 335 and 415 nm, and was found to be almost insensitive to the pH of the medium between 4.6 and 9. Transaminase B is active on phenylalanine as well, although the reaction between L-phenylalanine and alpha-ketoglutarate is about 50 to 100 times slower than the analogous reaction using L-valine as an aminoacid. Three sets of data show that the phenylalanine aminotransferase activity associated with transaminase B is not an artefact due to a protein contaminant. 1) Activities displayed toward phenylalanine and valine cannot be resolved by different methods, including chromatography, gel electrophoresis, and electrofucussing. 2) The absorption spectrum of the enzyme is as strongly modified by phenylalanine as by valine. 3) A ketoglutarate-free reaction between phenylalanine, tyrosine or typtophane and an aliphatic alpha-ketoacid is catalyzed by the pure enzyme and follows a mechanism belonging to the usual ping pong type. The possible significance of this reaction as a regulatory device in the cell metabolism is briefly discussed.

Phosphorylation of purified mitochondrial cytochromes P-450 (cholesterol desmolase and 11 beta-hydroxylase) from bovine adrenal cortex.

Two key steroidogenic mitochondrial cytochromes P-450 (cholesterol side-chain cleavage (scc) and 11 beta-hydroxylation (11 beta)) were purified from bovine adrenal cortex and examined as potential phosphorylatable substrates using purified cAMP-dependent protein kinase subunit (C) and A type (CKA) and G type (CKG) cAMP-independent casein kinases. Of the two cytochromes P-450, only P-450 11 beta was able to incorporate phosphate from ATP in the presence of C (Km = 7.5 microM), whereas CKA and CKG were ineffective. Phosphorylation of P-450 11 beta (maximum incorporation of 1 mole of 32P per mole of cytochrome, only on serine residues) did not modify the enzymatic activity of an 11 beta-hydroxylation system reconstituted in vitro from purified components, when adrenodoxin was in excess in the reaction. However, kinetic studies showed that P-450 11 beta phosphorylation strikingly increases the P-450 11 beta-adrenodoxin affinity in a phosphorylation-dependent manner. This would result in a net increase in 11 beta-hydroxylase activity under in vivo conditions where adrenodoxin availability is limited. Possible significance of these observations in the regulation of differentiated adrenocortical functions remains to be further examined.

Predicting the risk of cystic fibrosis with abnormal ultrasound signs of fetal bowel: results of a French molecular collaborative study based on 641 prospective cases.

Hyperechogenic fetal bowel is prenatally detected by ultrasound during the second trimester of pregnancy in 0.1-1.8% of fetuses. It has been described as a normal variant but has often been associated with severe diseases, notably cystic fibrosis (CF). The aim of our study was to determine the risk of CF in a prospective study of 641 fetuses with ultrasonographically abnormal fetal bowel and the residual risk when only one mutation is detected in the fetus. Fetal cells and/or parental blood cells were screened for CFTR mutations. Two screening steps were used, the first covering the mutations most frequently observed in French CF patients (mutation detection rate of 70-90%) and, when a CF mutation was detected, a DGGE-sequencing strategy. We observed a 3.1% risk of CF when a digestive tract anomaly was prenatally observed at routine ultrasound examination. The risk was higher when hyperechogenicity was associated with bowel dilatation (5/29; 17%) or with the absence of gall bladder (2/8; 25%). The residual risk of CF was 11% when only one CF mutation was detected by the first screening step, thereby justifying in-depth screening. Mutations associated with severe CF (DeltaF508 mutation) were more frequently observed in these ultrasonographically and prenatally detected CF cases. However, the frequency of heterozygous cases was that observed in the normal population, which demonstrates that heterozygous carriers of CF mutations are not at increased risk for hyperechogenic bowel. In conclusion, fetal bowel anomalies indicate a risk of severe cystic fibrosis and justify careful CFTR molecular analysis.

[Genetic of diseases by abnormal functioning of the skeletal muscle-calcium releasing complex]. / Génétique des pathologies associées à un dysfonctionnement du complexe de mobilisation calcique du muscle squelettique.

Myoplasmic calcium homeostasis is an essential feature of skeletal muscle contraction. The calcium mobilisation complex (CMC) located at the level of the triadic junction plays a major role for the regulation of calcium fluxes between extra-cellular, cytoplasmic and intra-cellular compartments. The ryanodine receptor type I (RYR1), which is located at the level of the terminal cisternae of the sarcoplasmic reticulum is a key component of the CMC. RYR1 allow the release into the myoplasm of the intralumenal stores of calcium. RYR1 interacts with other proteins: DiHydroPyridine Receptor, triadin, calsequestrin, FKBP12, calmodulin. Malignant hyperthermia (MHS) and congenital core myopathies have been associated with a dysfunction of the CMC. MHS is an autosomic dominant pharmacogenetic disease. The MH crisis is induced by exposure of the predisposed patients to halogenated volatile anaesthetics. MHS is characterised by a genetic heterogeneity and two genes, RYR1 and CACNA1S, have been associated so far with the disease. Mutations in the RYR1 gene have been recently associated with heat stroke, a related syndrome. Central Core Disease (CCD) and Multi minicore Disease (MmD) are congenital myopathies presenting with clinical variability and characterized by the presence of specific although heterogeneous muscle histological features: the cores. Clinical boundaries between the two diseases may overlap and the specific diagnosis is often based on the nature of the cores. These diseases show genetic heterogeneity with both autosomic dominant and recessive mode of inheritance and mutations in the SEPN1, RYR1, ACTA1, TPM3 genes have been reported. Mutations associated with MHS were mainly identified into 2 regions of the N-terminal part of RYR1. Functional role of these two domains is still unclear. Mutations responsible for congenital myopathies mainly mapped to the C terminal region of RYR1 that form the transmembrane calcium channel. Functional studies of the RYR1 mutations have shown that MHS mutations were mainly associated with an alteration of the calcium fluxes in response to caffeine or halothane while CCD mutations would result in a leaky RYR1 channel or would alter the Excitation-Contraction coupling at the level of the CMC.

[Biology of malignant hyperthermia: a disease of the calcium channels of the skeletal muscle]. / Biologie de l'hyperthermie maligne: une maladie des canaux calciques dumuscle squelettique.

Malignant hyperthermia susceptibility (MHS), a skeletal muscle disorder, is mostly inherited as an autosomal dominant trait. Exposure of susceptible individuals to volatile halogenated anaesthetics can lead to a MH episode resulting in irreversible tissue damages or to the patient's death if not immediately reversed by dantrolene treatment. A MH episode is characterised by a combination of hyperthermia, skeletal muscle rigidity and hypermetabolism. Porcine stress syndrome has proved to be a valuable model for physiopathological studies of MHS. Malignant hyperthermia syndrome is associated with a failure of the calcium homeostasis in muscular fibres. Dysfunction of the calcium channels: the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), which are involved in the release of the Ca2+ stored in sarcoplasmic reticulum has been clearly demonstrated. A biochemical test based on the analysis of the in vitro contracture response of muscular fibres to caffeine and halothane was developed to define the MHS status of patients. Although the genetic analysis of MHS has beneficiated from recent progresses, genetic testing is still far to answer to all testing situations. If in swine, hyperthermia syndrome was always associated with a unique mutation of the RyR1 gene, genetic analysis is far more complicated in human: i) more than 20 different MHS mutations in the RyR1 gene have been described; ii) a mutation of the gene encoding the dihydropyridine receptor has been identified; iii) 4 other potential MHS loci have been reported.

Congenital myopathy with focal loss of cross-striations revisited.

In 1977 Wijngaarden et al. reported a Dutch family with a congenital myopathy characterized by external ophthalmoplegia and a remarkable histological feature, focal loss of cross-striations. A small number of other families with similar clinical and pathological features led to the consideration of this congenital myopathy as a distinct entity. Here we present more than 30years of follow-up from the Dutch family and report recently identified compound heterozygous mutations in the skeletal muscle ryanodine receptor (RYR1) gene, c.10627-2A>G and p.Arg3539His (c.10616G>A). Focal loss of cross-striations on muscle biopsy is another histopathological feature that should raise the possibility of RYR1 involvement.

Can bubble columns be an alternative to fibrous filters for nanoparticles collection?

The most effective and widely used dedusting techniques to separate nanoparticles of a carrier fluid are fibrous media. The main problem is the clogging of the filter that induces a pressure drop increase over time and thus requires a regular cleaning of the media (or its replacement). Following these observations, this study proposes to investigate the potential of bubble columns for nanoparticles collection. Despite collection efficiencies lower than those of fibrous filters, experimental results show that bubble columns present likely performances for the collection of nanoparticles and have collection efficiency even more important when the liquid height is high and bubbling orifices have low diameters. Experiments have also revealed the presence of a most penetrating particle size for a particle diameter range between 10 and 30 nm. The model developed in this article highlights a good agreement between the theoretical collection efficiency by Brownian diffusion and experimental collection efficiencies for particles lower than 20 nm. Nevertheless, the modelling may be extended to other collection mechanisms in order to explain the collection efficiency increase for particles higher than 20 nm and to confirm or infirm that electrostatic effects can be the cause of this efficiency increase.