Molecular characterization of a Streptococcus gallolyticus genomic island encoding a pilus involved in endocarditis.

Background. Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. Methods. S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. Results. We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. Conclusions. This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

Encoding dysfunctions in a dynamic-static paradigm for visuospatial working memory in first-episode psychosis patients: a 2-year follow-up study.

Aim: To investigate static and dynamic visuospatial working memory (VSWM) processes in first-episode psychosis (FEP) patients and explore the validity of such measures as specific trait markers of schizophrenia. Methods: Twenty FEP patients and 20 age-, sex-, laterality- and education-matched controls carried out a dynamic and static VSWM paradigm. At 2-year follow up 13 patients met Diagnostic and Statistical Manual (of Mental Health Disorders) - Fourth Edition (DSM-IV) criteria for schizophrenia, 1 for bipolar disorder, 1 for brief psychotic episode and 5 for schizotypal personality disorder. Results: Compared with controls, the 20 FEP patients showed severe impairment in the dynamic VSWM condition but much less impairment in the static condition. No specific bias in stimulus selection was detected in the two tasks. Two-year follow-up evaluations suggested poorer baseline scores on the dynamic task clearly differentiated the 13 FEP patients who developed schizophrenia from the seven who did not. Conclusions: Results suggest deficits in VSWM in FEP patients. Specific exploratory analyses further suggest that deficit in monitoring-manipulation VSWM processes, especially involved in our dynamic VSWM task, can be a reliable marker of schizophrenia.

Expression of the gene encoding the high-Km glucose transporter 2 by the early postimplantation mouse embryo is essential for neural tube defects associated with diabetic embryopathy.

AIMS/HYPOTHESIS: Excess glucose transport to embryos during diabetic pregnancy causes congenital malformations. The early postimplantation embryo expresses the gene encoding the high-Km GLUT2 (also known as SLC2A2) glucose transporter. The hypothesis tested here is that high-Km glucose transport by GLUT2 causes malformations resulting from maternal hyperglycaemia during diabetic pregnancy. MATERIALS AND METHODS: Glut2 mRNA was assayed by RT-PCR. The Km of embryo glucose transport was determined by measuring 0.5-20 mmol/l 2-deoxy[3H]glucose transport. To test whether the GLUT2 transporter is required for neural tube defects resulting from maternal hyperglycaemia, Glut2+/- mice were crossed and transient hyperglycaemia was induced by glucose injection on day 7.5 of pregnancy. Embryos were recovered on day 10.5, and the incidence of neural tube defects in wild-type, Glut2+/- and Glut2-/- embryos was scored. RESULTS: Early postimplantation embryos expressed Glut2, and expression was unaffected by maternal diabetes. Moreover, glucose transport by these embryos showed Michaelis-Menten kinetics of 16.19 mmol/l, consistent with transport mediated by GLUT2. In pregnancies made hyperglycaemic on day 7.5, neural tube defects were significantly increased in wild-type embryos, but Glut2+/- embryos were partially protected from neural tube defects, and Glut2-/- embryos were completely protected from these defects. The frequency of occurrence of wild-type, Glut2+/- and Glut2-/- embryos suggests that the presence of Glut2 alleles confers a survival advantage in embryos before day 10.5. CONCLUSIONS/INTERPRETATIONS: High-Km glucose transport by the GLUT2 glucose transporter during organogenesis is responsible for the embryopathic effects of maternal diabetes.

Practical signal-to-noise ratio quantification for sensitivity encoding: Application to coronary MR angiography.

Purpose: To develop and evaluate a practical method for the quantification of signal-to-noise ratio (SNR) on coronary MR angiograms (MRA) acquired with parallel imaging.Materials and Methods: To quantify the spatially varying noise due to parallel imaging reconstruction, a new method has been implemented incorporating image data acquisition followed by a fast noise scan during which radio-frequency pulses, cardiac triggering and navigator gating are disabled. The performance of this method was evaluated in a phantom study where SNR measurements were compared with those of a reference standard (multiple repetitions). Subsequently, SNR of myocardium and posterior skeletal muscle was determined on in vivo human coronary MRA.Results: In a phantom, the SNR measured using the proposed method deviated less than 10.1% from the reference method for small geometry factors (<= 2). In vivo, the noise scan for a 10 min coronary MRA acquisition was acquired in 30 s. Higher signal and lower SNR, due to spatially varying noise, were found in myocardium compared with posterior skeletal muscle.Conclusion: SNR quantification based on a fast noise scan is a validated and easy-to-use method when applied to three-dimensional coronary MRA obtained with parallel imaging as long as the geometry factor remains low.

Autosomal Recessive Posterior Microphthalmos/Nanophthalmos is Caused by Loss-of-function Mutations in LOC646960, a Novel Gene Encoding a Trypsin-like Serine Protease

Purpose: Posterior microphthalmos (MCOP)/nanophthalmos (NNO) is a developmental anomaly characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal recessive form (arMCOP). The gene mutated in arMCOP is not yet known.Methods: Genetic mapping by linkage analysis using microsatellite and single nucleotide polymorphisms, mutation analysis by PCR and sequencing, molecular modellingResults: Having refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in Faroese families, we detected 3 mutations in a novel gene, LOC646960: Patients of 10 different Faroese families were either homozygous (n=22) for c.926G>C (p.Trp309Ser) or compound heterozygous (n=6) for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in patients with arNNO from a Tunisian family. In two unrelated patients with MCOP, no LOC646960 mutation was found. LOC646960 is expressed in the human adult retina and RPE. The expression of the mouse homologue in the eye can be first detected at E17 and is highest in adults. The predicted protein is a 603 amino acid long secreted trypsin-like serine peptidase. c.1066dupC should result in a functional null allele. Molecular modelling of the p.Trp309Ser mutant suggests that both affinity and reactivity of the enzyme towards in vivo substrates are substantially reduced.Conclusions: Postnatal growth of the eye is important for proper development of the refractive components (emmetropization), and is mainly due to elongation of the posterior segment from 10-11 mm at birth to 15-16 mm at the age of 13 years. Optical defocus leads to changes in axial length by moving the retina towards the image plane. arMCOP may theoretically be explained, in line with the expression pattern of LOC646960, by a postnatal growth retardation of the posterior segment.

Use of 2D Sensitivity Encoding for Slow-Infusion Contrast-Enhanced Isotropic 3-T Whole-Heart Coronary MR Angiography.

OBJECTIVE. The purpose of this study was to improve the blood-pool signal-to-noise ratio (SNR) and blood-myocardium contrast-to-noise ratio (CNR) of slow-infusion 3-T whole-heart coronary MR angiography (MRA).SUBJECTS AND METHODS. In 2D sensitivity encoding (SENSE), the number of acquired k-space lines is reduced, allowing less radiofrequency excitation per cardiac cycle and a longer TR. The former can be exploited for signal enhancement with a higher radiofrequency excitation angle, and the latter leads to noise reduction due to lower data-sampling bandwidth. Both effects contribute to SNR gain in coronary MRA when spatial and temporal resolution and acquisition time remain identical. Numeric simulation was performed to select the optimal 2D SENSE pulse sequence parameters and predict the SNR gain. Eleven patients underwent conventional unenhanced and the proposed 2D SENSE contrast-enhanced coronary MRA acquisition. Blood-pool SNR, blood-myocardium CNR, visible vessel length, vessel sharpness, and number of side branches were evaluated.RESULTS. Consistent with the numeric simulation, using 2D SENSE in contrast-enhanced coronary MRA resulted in significant improvement in aortic blood-pool SNR (unenhanced vs contrast-enhanced, 37.5 +/- 14.7 vs 121.3 +/- 44.0; p < 0.05) and CNR (14.4 +/- 6.9 vs 101.5 +/- 40.8; p < 0.05) in the patient sample. A longer length of left anterior descending coronary artery was visualized, but vessel sharpness, coronary artery coverage, and image quality score were not improved with the proposed approach.CONCLUSION. In combination with contrast administration, 2D SENSE was found effective in improving SNR and CNR in 3-T whole-heart coronary MRA. Further investigation of cardiac motion compensation is necessary to exploit the SNR and CNR advantages and to achieve submillimeter spatial resolution.

Local ocular immunomodulation resulting from electrotransfer of plasmid encoding soluble TNF receptors in the ciliary muscle.

PURPOSE: Plasmid electrotransfer in the ciliary muscle allows the sustained release of therapeutic proteins within the eye. The aim of this study was to evaluate whether the ocular production of TNF-alpha soluble receptor, using this nonviral gene therapy method, could have a beneficial local effect in a model of experimental autoimmune uveoretinitis (EAU). METHODS: Injection of a plasmid encoding a TNF-alpha p55 receptor (30 microg) in the ciliary muscle, combined with electrotransfer (200 V/cm), was carried out in Lewis rat eyes 4 days before the induction of EAU by S-antigen. Control eyes received naked plasmid electrotransfer or simple injection of the therapeutic plasmid. The disease was evaluated clinically and histologically. Cytokines and chemokines were analyzed in the ocular media by multiplex assay performed 15 and 21 days after immunization. RESULTS: Ocular TNF-alpha blockade, resulting from the local secretion of soluble receptors, was associated with delayed and significantly less severe uveitis, together with a reduction of the retinal damages. Compared with the controls, treated eyes showed significantly lower levels of IL-1beta and MCP1, higher levels of IL-13 and IL-4, and reduced NOS-2 expression in infiltrating cells. Treatment did not influence TNF-alpha levels in inguinal lymph nodes. CONCLUSIONS: Taken together, these results indicate that local immunomodulation was achieved and that no systemic adverse effects of TNF-alpha blockade observed after systemic injection of TNF-alpha inhibitors should be expected.

A Mutation in CALM1 Encoding Calmodulin in Familial Idiopathic Ventricular Fibrillation in Childhood and Adolescence.

OBJECTIVES: This study aimed to identify the genetic defect in a family with idiopathic ventricular fibrillation (IVF) manifesting in childhood and adolescence. BACKGROUND: Although sudden cardiac death in the young is rare, it frequently presents as the first clinical manifestation of an underlying inherited arrhythmia syndrome. Gene discovery for IVF is important as it enables the identification of individuals at risk, because except for arrhythmia, IVF does not manifest with identifiable clinical abnormalities. METHODS: Exome sequencing was carried out on 2 family members who were both successfully resuscitated from a cardiac arrest. RESULTS: We characterized a family presenting with a history of ventricular fibrillation (VF) and sudden death without electrocardiographic or echocardiographic abnormalities at rest. Two siblings died suddenly at the ages of 9 and 10 years, and another 2 were resuscitated from out-of-hospital cardiac arrest with documented VF at ages 10 and 16 years, respectively. Exome sequencing identified a missense mutation affecting a highly conserved residue (p.F90L) in the CALM1 gene encoding calmodulin. This mutation was also carried by 1 of the siblings who died suddenly, from whom DNA was available. The mutation was present in the mother and in another sibling, both asymptomatic but displaying a marginally prolonged QT interval during exercise. CONCLUSIONS: We identified a mutation in CALM1 underlying IVF manifesting in childhood and adolescence. The causality of the mutation is supported by previous studies demonstrating that F90 mediates the direct interaction of CaM with target peptides. Our approach highlights the utility of exome sequencing in uncovering the genetic defect even in families with a small number of affected individuals.

Real-time MR imaging of myocardial regional function using strain-encoding (SENC) with tissue through-plane motion tracking.

PURPOSE: To implement real-time myocardial strain-encoding (SENC) imaging in combination with tracking the tissue displacement in the through-plane direction. MATERIALS AND METHODS: SENC imaging was combined with the slice-following technique by implementing three-dimensional (3D) selective excitation. Certain adjustments were implemented to reduce scan time to one heartbeat. A total of 10 volunteers and five pigs were scanned on a 3T MRI scanner. Spatial modulation of magnetization (SPAMM)-tagged images were acquired on planes orthogonal to the SENC planes for comparison. Myocardial infarction (MI) was induced in two pigs and the resulting SENC images were compared to standard delayed-enhancement (DE) images. RESULTS: The strain values computed from SENC imaging with slice-following showed significant difference from those acquired without slice-following, especially during systole (P < 0.01). The strain curves computed from the SENC images with and without slice-following were similar to those computed from the orthogonal SPAMM images, with and without, respectively, tracking the tag line displacement in the strain direction. The resulting SENC images showed good agreement with the DE images in identifying MI in infarcted pigs. CONCLUSION: Correction of through-plane motion in real-time cardiac functional imaging is feasible using slice-following. The strain measurements are more accurate than conventional SENC measurements in humans and animals, as validated with conventional MRI tagging.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.

Simulation-based evaluation of susceptibility distortion correction methods in diffusion MRI for connectivity analysis

Connectivity analysis on diffusion MRI data of the whole- brain suffers from distortions caused by the standard echo- planar imaging acquisition strategies. These images show characteristic geometrical deformations and signal destruction that are an important drawback limiting the success of tractography algorithms. Several retrospective correction techniques are readily available. In this work, we use a digital phantom designed for the evaluation of connectivity pipelines. We subject the phantom to a âeurooetheoretically correctâeuro and plausible deformation that resembles the artifact under investigation. We correct data back, with three standard methodologies (namely fieldmap-based, reversed encoding-based, and registration- based). Finally, we rank the methods based on their geometrical accuracy, the dropout compensation, and their impact on the resulting connectivity matrices.

Exploration of the visual system : Part 2. In vivo analysis methods : virtual-reality optomotor system, fundus examination and fluorescent angiography

The mouse has emerged as an animal model for many diseases. At IRO, we have used this animal to understand the development of many eye diseases and treatment of some of them. Precise evaluation of vision is a prerequisite for both these approaches. In this unit we describe three ways to measure vision: testing the optokinetic response, and evaluating the fundus by direct observation and by fluorescent angiography.

Liquid chromatography-tandem mass spectrometry (LC/APCI-MS/MS) methods for the quantification of captan and folpet phthalimide metabolites in human plasma and urine.

Captan and folpet are fungicides largely used in agriculture. They have similar chemical structures, except that folpet has an aromatic ring unlike captan. Their half-lives in blood are very short, given that they are readily broken down to tetrahydrophthalimide (THPI) and phthalimide (PI), respectively. Few authors measured these biomarkers in plasma or urine, and analysis was conducted either by gas chromatography coupled to mass spectrometry or liquid chromatography with UV detection. The objective of this study was thus to develop simple, sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS) methods to quantify both THPI and PI in human plasma and urine. Briefly, deuterated THPI was added as an internal standard and purification was performed by solid-phase extraction followed by LC/APCI-MS/MS analysis in negative ion mode for both compounds. Validation of the methods was conducted using spiked blank plasma and urine samples at concentrations ranging from 1 to 250 μg/L and 1 to 50 μg/L, respectively, along with samples of volunteers and workers exposed to captan or folpet. The methods showed a good linearity (R (2) > 0.99), recovery (on average 90% for THPI and 75% for PI), intra- and inter-day precision (RSD, <15%) and accuracy (<20%), and stability. The limit of detection was 0.58 μg/L in urine and 1.47 μg/L in plasma for THPI and 1.14 and 2.17 μg/L, respectively, for PI. The described methods proved to be accurate and suitable to determine the toxicokinetics of both metabolites in human plasma and urine.

New methods for detecting antibiotic resistant bacteria and preventing their spread

Methicillin resistant Staphylococcus aureus (MRSA) bacteria have emerged in the early 1980's in numerous health care institutions around the world. The main transmission mechanism within hospitals and healthcare facilities is through the hands of health care workers. Resistant to several antibiotics, the MRSA is one of the most feared pathogens in the hospital setting since it is very difficult to eradicate with the standard treatments. There are still a limited number of anti-MRSA antibiotics but the first cases of resistance to these compounds have already been reported and their frequency is likely to increase in the coming years. Every year, the MRSA infections result in major human and financial costs, due to the high associated mortality and expenses related to the required care. Measures towards a faster detection of resistant bacteria and establishment of appropriate antibiotic treatment parameters are fundamental. Also as part as infection prevention, diminution of bacteria present on the commonly touched surfaces could also limit the spread and selection of antibiotic resistant bacteria. During my thesis, projects were developed around MRSA and antibiotic resistance investigation using innovative technologies. The thesis was subdivided in three main parts with the use of atomic force microscopy AFM for antibiotic resistance detection in part 1, the importance of the bacterial inoculum size in the selection of antibiotic resistance in part 2 and the testing of antimicrobial surfaces creating by sputtering copper onto polyester in part 3. In part 1 the AFM was used two different ways, first for the measurement of stiffness (elasticity) of bacteria and second as a nanosensor for antibiotic susceptibility testing. The stiffness of MRSA with different susceptibility profiles to vancomycin was investigated using the stiffness tomography mode of the AFM and results have demonstrated and increased stiffness in the vancomycin resistant strains that also paralleled with increased thickness of the bacterial cell wall. Parts of the AFM were also used to build a new antibiotic susceptibility-testing device. This nano sensor was able to measure vibrations emitted from living bacteria that ceased definitively upon antibiotic exposure to which they were susceptible but restarted after antibiotic removal to which they were resistant, allowing in a matter of minute the assessment of antibiotic susceptibility determination. In part 2 the inoculum effect (IE) of vancomycin, daptomycin and linezolid and its importance in antibiotic resistance selection was investigated with MRSA during a 15 days of cycling experiment. Results indicated that a high bacterial inoculum and a prolonged antibiotic exposure were two key factors in the in vitro antibiotic resistance selection in MRSA and should be taken into consideration when choosing the drug treatment. Finally in part 3 bactericidal textile surfaces were investigated against MRSA. Polyesters coated after 160 seconds of copper sputtering have demonstrated a high bactericidal activity reducing the bacterial load of at least 3 logio after one hour of contact. -- Au cours des dernières décennies, des bactéries multirésistantes aux antibiotiques (BMR) ont émergé dans les hôpitaux du monde entier. Depuis lors, le nombre de BMR et la prévalence des infections liées aux soins (IAS) continuent de croître et sont associés à une augmentation des taux de morbidité et de mortalité ainsi qu'à des coûts élevés. De plus, le nombre de résistance à différentes classes d'antibiotiques a également augmenté parmi les BMR, limitant ainsi les options thérapeutiques disponibles lorsqu'elles ont liées a des infections. Des mesures visant une détection plus rapide des bactéries résistantes ainsi que l'établissement des paramètres de traitement antibiotiques adéquats sont primordiales lors d'infections déjà présentes. Dans une optique de prévention, la diminution des bactéries présentes sur les surfaces communément touchées pourrait aussi freiner la dissémination et l'évolution des bactéries résistantes. Durant ma thèse, différents projets incluant des nouvelles technologies et évoluant autour de la résistance antibiotique ont été traités. Des nouvelles technologies telles que le microscope à force atomique (AFM) et la pulvérisation cathodique de cuivre (PCC) ont été utilisées, et le Staphylococcus aureus résistant à la méticilline (SARM) a été la principale BMR étudiée. Deux grandes lignes de recherche ont été développées; la première visant à détecter la résistance antibiotique plus rapidement avec l'AFM et la seconde visant à prévenir la dissémination des BMR avec des surfaces crées grâce à la PCC. L'AFM a tout d'abord été utilisé en tant que microscope à sonde locale afin d'investiguer la résistance à la vancomycine chez les SARMs. Les résultats ont démontré que la rigidité de la paroi augmentait avec la résistance à la vancomycine et que celle-ci corrélait aussi avec une augmentation de l'épaisseur des parois, vérifiée grâce à la microscopie électronique. Des parties d'un AFM ont été ensuite utilisées afin de créer un nouveau dispositif de test de sensibilité aux antibiotiques, un nanocapteur. Ce nanocapteur mesure des vibrations produites par les bactéries vivantes. Après l'ajout d'antibiotique, les vibrations cessent définitivement chez les bactéries sensibles à l'antibiotique. En revanche pour les bactéries résistantes, les vibrations reprennent après le retrait de l'antibiotique dans le milieu permettant ainsi, en l'espace de minutes de détecter la sensibilité de la bactérie à un antibiotique. La PCC a été utilisée afin de créer des surfaces bactéricides pour la prévention de la viabilité des BMR sur des surfaces inertes. Des polyesters finement recouverts de cuivre (Cu), connu pour ses propriétés bactéricides, ont été produits et testés contre des SARMs. Une méthode de détection de viabilité des bactéries sur ces surfaces a été mise au point, et les polyesters obtenus après 160 secondes de pulvérisation au Cu ont démontré une excellente activité bactéricide, diminuant la charge bactérienne d'au moins 3 logio après une heure de contact. En conclusion, l'utilisation de nouvelles technologies nous a permis d'évoluer vers de méthodes de détection de la résistance antibiotique plus rapides ainsi que vers le développement d'un nouveau type de surface bactéricide, dans le but d'améliorer le diagnostic et la gestion des BMR.