Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing

BACKGROUND: Sequencing based mutation screening assays of genes encompassing large numbers of exons could be substantially optimized by multiplex PCR, which enables simultaneous amplification of many targets in one reaction. In the present study, a multiplex PCR protocol originally developed for fragment analysis was evaluated for sequencing based mutation screening of the ornithine transcarbamylase (OTC) and the medium-chain acyl-CoA dehydrogenase (MCAD) genes. METHODS: Single exon and multiplex PCR protocols were applied to generate PCR templates for subsequent DNA sequencing of all exons of the OTC and the MCAD genes. For each PCR protocol and using the same DNA samples, 66 OTC and 98 MCAD sequence reads were generated. The sequences derived from the two different PCR methods were compared at the level of individual signal-to-noise ratios of the four bases and the proportion of high-quality base-signals. RESULTS: The single exon and the multiplex PCR protocol gave qualitatively comparable results for the two genes. CONCLUSIONS: Many existing sequencing based mutation analysis protocols may be easily optimized with the proposed method, since the multiplex PCR protocol was successfully applied without any re-design of the PCR primers and other optimization steps for generating sequencing templates for the OTC and MCAD genes, respectively.

A three-dimensional histological atlas of the human basal ganglia. II. Atlas deformation strategy and evaluation in deep brain stimulation for Parkinson disease

OBJECT: The localization of any given target in the brain has become a challenging issue because of the increased use of deep brain stimulation to treat Parkinson disease, dystonia, and nonmotor diseases (for example, Tourette syndrome, obsessive compulsive disorders, and depression). The aim of this study was to develop an automated method of adapting an atlas of the human basal ganglia to the brains of individual patients. METHODS: Magnetic resonance images of the brain specimen were obtained before extraction from the skull and histological processing. Adaptation of the atlas to individual patient anatomy was performed by reshaping the atlas MR images to the images obtained in the individual patient using a hierarchical registration applied to a region of interest centered on the basal ganglia, and then applying the reshaping matrix to the atlas surfaces. RESULTS: Results were evaluated by direct visual inspection of the structures visible on MR images and atlas anatomy, by comparison with electrophysiological intraoperative data, and with previous atlas studies in patients with Parkinson disease. The method was both robust and accurate, never failing to provide an anatomically reliable atlas to patient registration. The registration obtained did not exceed a 1-mm mismatch with the electrophysiological signatures in the region of the subthalamic nucleus. CONCLUSIONS: This registration method applied to the basal ganglia atlas forms a powerful and reliable method for determining deep brain stimulation targets within the basal ganglia of individual patients.

The Mycoplasma conjunctivae genome sequencing, annotation and analysis

BACKGROUND: The mollicute Mycoplasma conjunctivae is the etiological agent leading to infectious keratoconjunctivitis (IKC) in domestic sheep and wild caprinae. Although this pathogen is relatively benign for domestic animals treated by antibiotics, it can lead wild animals to blindness and death. This is a major cause of death in the protected species in the Alps (e.g., Capra ibex, Rupicapra rupicapra). METHODS: The genome was sequenced using a combined technique of GS-FLX (454) and Sanger sequencing, and annotated by an automatic pipeline that we designed using several tools interconnected via PERL scripts. The resulting annotations are stored in a MySQL database. RESULTS: The annotated sequence is deposited in the EMBL database (FM864216) and uploaded into the mollicutes database MolliGen http://cbi.labri.fr/outils/molligen/ allowing for comparative genomics. CONCLUSION: We show that our automatic pipeline allows for annotating a complete mycoplasma genome and present several examples of analysis in search for biological targets (e.g., pathogenic proteins).

A deep dig--hindsight on Holocene vegetation composition from ancient environmental DNA

Want a glimpse at past vegetation? Studying pollen and other plant remains, which are preserved for example in lake sediments or mires for thousands of years, allows us to document regional occurrences of plant species over radiocarbon-dated time series. Such vegetation reconstructions derived from optical analyses of fossil samples are inherently incomplete because they only comprise taxa that contribute sufficient amounts of pollen, spores, macrofossil or other evidences. To complement optical analyses for paleoecological inference, molecular markers applied to ancient DNA (aDNA) may help in disclosing information hitherto inaccessible to biologists. Parducci et al. (2013) targeted aDNA from sediment cores of two lakes in the Scandes Mountains with generic primers in a meta-barcoding approach. When compared to palynological records from the same cores, respective taxon lists show remarkable differences in their compositions, but also in quantitative representation and in taxonomic resolution similar to a previous study (Jørgensen et al. 2012). While not free of assumptions that need critical and robust testing, notably the question of possible contamination, this study provides thrilling prospects to improve our knowledge about past vegetation composition, but also other organismic groups, stored as a biological treasure in the ground.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Phylogenetic analysis of Pasteurella multocida subspecies and molecular identification of feline P. multocida subsp. septica by 16S rRNA gene sequencing

Pasteurella multocida is commonly found in the oral cavity of cats and dogs. In humans it is known as an opportunistic pathogen after bites from these animals. Phenotypic identification of P. multocida based on biochemical reactions is often limited and usually only done on a species level, even though 3 subspecies are described. For molecular taxonomy and diagnostic purposes a phylogenetic analysis of the three subspecies of P. multocida based on their 16S rRNA (rrs) gene sequence was therefore carried out. We found P. multocida subsp. septica on a distinguished branch on the phylogenetic tree of Pasteurellaceae, due to a 1.5% divergence of its rrs gene compared to the two other, more closely related subspecies multocida and gallicida. This phylogenetic divergence can be used for the identification of P. multocida subsp. septica by rrs gene determination since they form a phylogenetically well isolated and defined group as shown with a set of feline isolates. Comparison to routine phenotypic identification shows the advantage of the sequence-based identification over conventional methods. It is therefore helpful for future unambiguous identification and molecular taxonomy of P. multocida as well as for epidemiological investigations.

Influence of deep sternal wound infection on long-term survival after cardiac surgery

BACKGROUND: This study aimed to investigate the influence of deep sternal wound infection on long-term survival following cardiac surgery. MATERIAL AND METHODS: In our institutional database we retrospectively evaluated medical records of 4732 adult patients who received open-heart surgery from January 1995 through December 2005. The predictive factors for DSWI were determined using logistic regression analysis. Then, each patient with deep sternal wound infection (DSWI) was matched with 2 controls without DSWI, according to the risk factors identified previously. After checking balance resulting from matching, short-term mortality was compared between groups using a paired test, and long-term survival was compared using Kaplan-Meier analysis and a Cox proportional hazard model. RESULTS: Overall, 4732 records were analyzed. The mean age of the investigated population was 69.3±12.8 years. DSWI occurred in 74 (1.56%) patients. Significant independent predictive factors for deep sternal infections were active smoking (OR 2.19, CI95 1.35-3.53, p=0.001), obesity (OR 1.96, CI95 1.20-3.21, p=0.007), and insulin-dependent diabetes mellitus (OR 2.09, CI95 1.05-10.06, p=0.016). Mean follow-up in the matched set was 125 months, IQR 99-162. After matching, in-hospital mortality was higher in the DSWI group (8.1% vs. 2.7% p=0.03), but DSWI was not an independent predictor of long-term survival (adjusted HR 1.5, CI95 0.7-3.2, p=0.33). CONCLUSIONS: The results presented in this report clearly show that post-sternotomy deep wound infection does not influence long-term survival in an adult general cardio-surgical patient population.