Sequencing of Lp-PLA2-encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations.

Elevated plasma levels of lipoprotein-associated phospholipase A(2) (Lp-PLA2) activity have been shown to be associated with increased risk of coronary heart disease and an inhibitor of this enzyme is under development for the treatment of that condition. A Val279Phe null allele in this gene, that may influence patient eligibility for treatment, is relatively common in East Asians but has not been observed in Europeans. We investigated the existence and functional effects of low frequency alleles in a Western European population by re-sequencing the exons of PLA2G7 in 2000 samples. In all, 19 non-synonymous single-nucleotide polymorphisms (nsSNPs) were found, 14 in fewer than four subjects (minor allele frequency <0.1%). Lp-PLA2 activity was significantly lower in rare nsSNP carriers compared with non-carriers (167.8±63.2 vs 204.6±41.8, P=0.01) and seven variants had enzyme activities consistent with a null allele. The cumulative frequency of these null alleles was 0.25%, so <1 in 10,000 Europeans would be expected to be homozygous, and thus not potentially benefit from treatment with an Lp-PLA2 inhibitor.

A connectome-based comparison of diffusion MRI schemes.

Diffusion MRI has evolved towards an important clinical diagnostic and research tool. Though clinical routine is using mainly diffusion weighted and tensor imaging approaches, Q-ball imaging and diffusion spectrum imaging techniques have become more widely available. They are frequently used in research-oriented investigations in particular those aiming at measuring brain network connectivity. In this work, we aim at assessing the dependency of connectivity measurements on various diffusion encoding schemes in combination with appropriate data modeling. We process and compare the structural connection matrices computed from several diffusion encoding schemes, including diffusion tensor imaging, q-ball imaging and high angular resolution schemes, such as diffusion spectrum imaging with a publically available processing pipeline for data reconstruction, tracking and visualization of diffusion MR imaging. The results indicate that the high angular resolution schemes maximize the number of obtained connections when applying identical processing strategies to the different diffusion schemes. Compared to the conventional diffusion tensor imaging, the added connectivity is mainly found for pathways in the 50-100mm range, corresponding to neighboring association fibers and long-range associative, striatal and commissural fiber pathways. The analysis of the major associative fiber tracts of the brain reveals striking differences between the applied diffusion schemes. More complex data modeling techniques (beyond tensor model) are recommended 1) if the tracts of interest run through large fiber crossings such as the centrum semi-ovale, or 2) if non-dominant fiber populations, e.g. the neighboring association fibers are the subject of investigation. An important finding of the study is that since the ground truth sensitivity and specificity is not known, the comparability between results arising from different strategies in data reconstruction and/or tracking becomes implausible to understand.

Long-term monitoring of post-stroke plasticity after transient cerebral ischemia in mice using in vivo and ex vivo diffusion tensor MRI.

WE USED A MURINE MODEL OF TRANSIENT FOCAL CEREBRAL ISCHEMIA TO STUDY: 1) in vivo DTI long-term temporal evolution of the apparent diffusion coefficient (ADC) and diffusion fractional anisotropy (FA) at days 4, 10, 15 and 21 after stroke 2) ex vivo distribution of a plasticity-related protein (GAP-43) and its relationship with the ex vivo DTI characteristics of the striato-thalamic pathway (21 days). All animals recovered motor function. In vivo ADC within the infarct was significantly increased after stroke. In the stroke group, GAP-43 expression and FA values were significantly higher in the ipsilateral (IL) striatum and contralateral (CL) hippocampus compared to the shams. DTI tractography showed fiber trajectories connecting the CL striatum to the stroke region, where increased GAP43 and FA were observed and fiber tracts from the CL striatum terminating in the IL hippocampus.Our data demonstrate that DTI changes parallel histological remodeling and recovery of function.

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.

Etude des gènes codant les protéines de gouttelettes lipidiques chez les patients avec la stéatose hépatique[Study of genes encoding proteins of lipid droplets in patients with fatty liver]

Introduction: La prévalence de la «non-alcoholic fatty liver disease (NAFLD)» dans les pays industrialisés augment de manière exponentielle. La NAFLD se développe d'une simple stéatose hépatique jusqu'à l'hépatite, puis à la cirrhose. De plus, la stéatose hépatique est fréquemment accompagnée par une résistance à l'insuline, une des causes principales du diabète. Les lipides intermédiaires, tels que céramides et diacylglycérols, ont été décrits comme induisant la résistance à l'insuline. Cependant, nous avons démontré dans notre modèle de stéatose hépatique, que les souris présentant une invalidation de la protéine «microsomal triglyceride transfer protein» (Mtpp) au niveau hépatique, ne développent pas de résistance à l'insuline. Ceci suggère fortement l'existence d'autres mécanismes susceptibles d'induire la résistance à l'insuline. Résultats: Grâce à une analyse de Microarray, nous avons observé une augmentation de l'expression des gènes «cell-death inducing DFFA-like effector c (CIDEC)», «lipid storage droplet protein 5 (LSDP5)» et «Bernardinelli-Seip congenital lipodystrophy 2 homolog (Seipin)» dans le foie des souris Mttp. Ces gènes ont récemment été identifiés comme des protéines localisées autour des gouttelettes lipidiques. Nous avons également constaté que la souris Mttp développe plutôt une microstéatose (petites gouttelettes lipidiques) qu'une macrostéatose qui est normalement observée chez les patients avec NAFLD. Nous avons étudié l'expression des gènes associés aux gouttelettes lipidiques chez les patients obèses avec stéatose hépatique, avec ou sans résistance à l'insuline. Comparés aux sujets sains sans stéatose hépatique, les patients avec la stéatose ont une expression significativement plus élevée. De manière intéressante, les patients avec résistance à l'insuline ont une diminution de ces expressions. Conclusion : Ces données suggèrent que les gènes des gouttelettes lipidiques sont impliqués dans le développement de la stéatose hépatique chez l'homme et peut-être contribue à la mise en place de la résistance à l'insuline.

Multiplication of an ancestral gene encoding secreted fungalysin preceded species differentiation in the dermatophytes Trichophyton and Microsporum.

Dermatophytes are human and animal pathogenic fungi which cause cutaneous infections and grow exclusively in the stratum corneum, nails and hair. In a culture medium containing soy proteins as sole nitrogen source a substantial proteolytic activity was secreted by Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. This proteolytic activity was 55-75 % inhibited by o-phenanthroline, attesting that metalloproteases were secreted by all three species. Using a consensus probe constructed on previously characterized genes encoding metalloproteases (MEP) of the M36 fungalysin family in Aspergillus fumigatus, Aspergillus oryzae and M. canis, a five-member MEP family was isolated from genomic libraries of T. rubrum, T. mentagrophytes and M. canis. A phylogenetic analysis of genomic and protein sequences revealed a robust tree consisting of five main clades, each of them including a MEP sequence type from each dermatophyte species. Each MEP type was remarkably conserved across species (72-97 % amino acid sequence identity). The tree topology clearly indicated that the multiplication of MEP genes in dermatophytes occurred prior to species divergence. In culture medium containing soy proteins as a sole nitrogen source secreted Meps accounted for 19-36 % of total secreted protein extracts; characterization of protein bands by proteolysis and mass spectrometry revealed that the three dermatophyte species secreted two Meps (Mep3 and Mep4) encoded by orthologous genes.

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.

Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens

Pseudomonas fluorescens CHA0 and the related strain Pf-5 are well-characterized representatives of rhizosphere bacteria that have the capacity to protect crop plants from fungal root diseases, mainly by releasing a variety of exoproducts that are toxic to plant pathogenic fungi. Here, we report that the two plant-beneficial pseudomonads also exhibit potent insecticidal activity. Anti-insect activity is linked to a novel genomic locus encoding a large protein toxin termed Fit (for P. fluorescensinsecticidal toxin) that is related to the insect toxin Mcf (Makes caterpillars floppy) of the entomopathogen Photorhabdus luminescens, a mutualist of insect-invading nematodes. When injected into the haemocoel, even low doses of P. fluorescens CHA0 or Pf-5 killed larvae of the tobacco hornworm Manduca sexta and the greater wax moth Galleria mellonella. In contrast, mutants of CHA0 or Pf-5 with deletions in the Fit toxin gene were significantly less virulent to the larvae. When expressed from an inducible promoter in a non-toxic Escherichia coli host, the Fit toxin gene was sufficient to render the bacterium toxic to both insect hosts. Our findings establish the Fit gene products of P. fluorescens CHA0 and Pf-5 as potent insect toxins that define previously unappreciated anti-insect properties of these plant-colonizing bacteria

Multi-slice three-dimensional myocardial strain tensor quantification using zHARP.

In this article we propose a novel method for calculating cardiac 3-D strain. The method requires the acquisition of myocardial short-axis (SA) slices only and produces the 3-D strain tensor at every point within every pair of slices. Three-dimensional displacement is calculated from SA slices using zHARP which is then used for calculating the local displacement gradient and thus the local strain tensor. There are three main advantages of this method. First, the 3-D strain tensor is calculated for every pixel without interpolation; this is unprecedented in cardiac MR imaging. Second, this method is fast, in part because there is no need to acquire long-axis (LA) slices. Third, the method is accurate because the 3-D displacement components are acquired simultaneously and therefore reduces motion artifacts without the need for registration. This article presents the theory of computing 3-D strain from two slices using zHARP, the imaging protocol, and both phantom and in-vivo validation.

Localization and fine structure of a vaccinia virus gene encoding an envelope antigen.

The major antigen on the envelope of extracellular vaccinia virus particles is a polypeptide with an apparent molecular weight of 37,000 (p37K; G. Hiller and K. Weber, J. Virol. 55:651-659, 1985). The gene encoding p37K was mapped in the vaccinia virus genome by hybrid selection of RNA followed by in vitro translation. p37K was then identified among the in vitro translation products by immunoprecipitation with a monoclonal antibody. The gene is located close to the right-hand end of the HindIII F fragment. The corresponding region of the DNA was sequenced, and an open reading frame encoding a polypeptide of 41,748 daltons was observed. The 5' end of the mRNA, as defined by nuclease S1 analysis, maps within only a few nucleotides of the translation initiation codon. Examination of the DNA sequence around the putative initiation site of transcription revealed a characteristic sequence, TAAATG, which includes the ATG translation initiation codon and which is conserved in all but one late gene so far analyzed. It is therefore likely that this sequence is an important regulatory signal for late gene expression in vaccinia virus.

Mutations in NDUFB11, Encoding a Complex I Component of the Mitochondrial Respiratory Chain, Cause Microphthalmia with Linear Skin Defects Syndrome.

Microphthalmia with linear skin defects (MLS) syndrome is an X-linked male-lethal disorder also known as MIDAS (microphthalmia, dermal aplasia, and sclerocornea). Additional clinical features include neurological and cardiac abnormalities. MLS syndrome is genetically heterogeneous given that heterozygous mutations in HCCS or COX7B have been identified in MLS-affected females. Both genes encode proteins involved in the structure and function of complexes III and IV, which form the terminal segment of the mitochondrial respiratory chain (MRC). However, not all individuals with MLS syndrome carry a mutation in either HCCS or COX7B. The majority of MLS-affected females have severe skewing of X chromosome inactivation, suggesting that mutations in HCCS, COX7B, and other as-yet-unidentified X-linked gene(s) cause selective loss of cells in which the mutated X chromosome is active. By applying whole-exome sequencing and filtering for X-chromosomal variants, we identified a de novo nonsense mutation in NDUFB11 (Xp11.23) in one female individual and a heterozygous 1-bp deletion in a second individual, her asymptomatic mother, and an affected aborted fetus of the subject's mother. NDUFB11 encodes one of 30 poorly characterized supernumerary subunits of NADH:ubiquinone oxidoreductase, known as complex I (cI), the first and largest enzyme of the MRC. By shRNA-mediated NDUFB11 knockdown in HeLa cells, we demonstrate that NDUFB11 is essential for cI assembly and activity as well as cell growth and survival. These results demonstrate that X-linked genetic defects leading to the complete inactivation of complex I, III, or IV underlie MLS syndrome. Our data reveal an unexpected role of cI dysfunction in a developmental phenotype, further underscoring the existence of a group of mitochondrial diseases associated with neurocutaneous manifestations.

Criblamydia sequanensis Harbors a Megaplasmid Encoding Arsenite Resistance.

Criblamydia sequanensis is an amoeba-resisting bacterium recently isolated from the Seine River. This Chlamydia-related bacterium harbors a genome of approximately 3 Mbp and a megaplasmid of 89,525 bp. The plasmid encodes several efflux systems and an operon for arsenite resistance. This first genome sequence within the Criblamydiaceae family enlarges our view on the evolution and the ecology of this important bacterial clade largely understudied so far.