Genomic sequencing of two isolates of Ralstonia solanacearum causing Sergipe facies and comparative analysis with Bugtok disease isolates

Ralstonia solanacearum is the causal agent of Moko disease in bananas, which in the state of Sergipe in northeastern Brazil causes “Sergipe facies”. This disease induces atypical symptoms similar to those of Bugtok disease in the Philippines. This study was conducted to sequence, assemble, and annotate the genomes of the Sergipe facies-causing isolates SFC and IBSBF2570 (sequevar IIA-53) and compare their genomes with two representative isolates causing Bugtok disease. The genomes were sequenced and assembled, resulting in lengths of 5.58 Mb (SFC) and 5.46 Mb (IBSBF2570) in 185 and 174 contigs, respectively. The isolates of Sergipe facies and Bugtok disease showed similarities in their gene contents. We identified 5,668 information clusters, 3,752 of which were shared by all genomes (core genes). Moreover, 3,585 single-copy genes were identified. Isolates causing Bugtok disease exclusively shared 266 more information clusters than the isolates causing Sergipe facies. These results suggest that Sergipe facies and Bugtok disease isolates show high genomic similarity. However, the similarity is even greater between the Bugtok disease isolates. This may be because of their longer period of interaction compared to Sergipe facies isolates.     

Tips and tricks for the assembly of a Corynebacterium pseudotuberculosis genome using a semiconductor sequencer

New sequencing platforms have enabled rapid decoding of complete prokaryotic genomes at relatively low cost. The Ion Torrent platform is an example of these technologies, characterized by lower coverage, generating challenges for the genome assembly. One particular problem is the lack of genomes that enable reference-based assembly, such as the one used in the present study, Corynebacterium pseudotuberculosis biovar equi, which causes high economic losses in the US equine industry. The quality treatment strategy incorporated into the assembly pipeline enabled a 16-fold greater use of the sequencing data obtained compared with traditional quality filter approaches. Data preprocessing prior to the de novo assembly enabled the use of known methodologies in the next-generation sequencing data assembly. Moreover, manual curation was proved to be essential for ensuring a quality assembly, which was validated by comparative genomics with other species of the genus Corynebacterium. The present study presents a modus operandi that enables a greater and better use of data obtained from semiconductor sequencing for obtaining the complete genome from a prokaryotic microorganism, C. pseudotuberculosis, which is not a traditional biological model such as Escherichia coli.     

Using High-Throughput Sequencing to Leverage Surveillance of Genetic Diversity and Oseltamivir Resistance: A Pilot Study during the 2009 Influenza A(H1N1) Pandemic

Background

Influenza viruses display a high mutation rate and complex evolutionary patterns. Next-generation sequencing (NGS) has been widely used for qualitative and semi-quantitative assessment of genetic diversity in complex biological samples. The “deep sequencing” approach, enabled by the enormous throughput of current NGS platforms, allows the identification of rare genetic viral variants in targeted genetic regions, but is usually limited to a small number of samples.

Methodology and Principal Findings

We designed a proof-of-principle study to test whether redistributing sequencing throughput from a high depth-small sample number towards a low depth-large sample number approach is feasible and contributes to influenza epidemiological surveillance. Using 454-Roche sequencing, we sequenced at a rather low depth, a 307 bp amplicon of the neuraminidase gene of the Influenza A(H1N1) pandemic (A(H1N1)pdm) virus from cDNA amplicons pooled in 48 barcoded libraries obtained from nasal swab samples of infected patients (n  =  299) taken from May to November, 2009 pandemic period in Mexico. This approach revealed that during the transition from the first (May-July) to second wave (September-November) of the pandemic, the initial genetic variants were replaced by the N248D mutation in the NA gene, and enabled the establishment of temporal and geographic associations with genetic diversity and the identification of mutations associated with oseltamivir resistance.

Conclusions

NGS sequencing of a short amplicon from the NA gene at low sequencing depth allowed genetic screening of a large number of samples, providing insights to viral genetic diversity dynamics and the identification of genetic variants associated with oseltamivir resistance. Further research is needed to explain the observed replacement of the genetic variants seen during the second wave. As sequencing throughput rises and library multiplexing and automation improves, we foresee that the approach presented here can be scaled up for global genetic surveillance of influenza and other infectious diseases.     

Treatment of children with high grade glioma with nimotuzumab: A 5-y institutional experience

Brain tumors are a major cause of cancer-related mortality in children. Overexpression of epidermal growth factor receptor (EGFR) is detected in pediatric brain tumors and receptor density appears to increase with tumor grading. Nimotuzumab is an IgG1 antibody that targets EGFR. Twenty-three children with high-grade glioma (HGG) were enrolled in an expanded access program in which nimotuzumab was administered alone or with radio-chemotherapy. The mean number of doses was 39. Nimotuzumab was well-tolerated and treatment with the antibody yielded a survival benefit: median survival time was 32.66 mo and the 2-y survival rate was 54.2%. This study demonstrated the feasibility of prolonged administration of nimotuzumab and showed preliminary evidence of clinical benefit in HGG patients with poor prognosis.     

Impact of a Partial Smoke-Free Legislation on Myocardial Infarction Incidence,Mortality and Case-Fatality in a Population-Based Registry: The REGICOR Study

Background and Objective

Coronary heart disease (CHD) is the leading cause of death, and smoking its strongest modifiable risk factor. Our aim was to determine the impact of the Spanish 2006 partial smoke-free legislation on acute myocardial infarction (AMI) incidence, hospitalization and mortality rates, and 28-day case-fatality in Girona, Spain.

Methods

Using a population-based registry (the REGICOR Study), we compared population incidence, hospitalization, and mortality rates, and 28-day case-fatality in the pre- and post-ban periods (2002–2005 and 2006–2008, respectively) by binomial regression analysis adjusted for confounding factors. We also analyzed the ban''s impact on the outcomes of interest using the AMI definitions of the American Heart Association (AHA)/European Society of Cardiology (ESC) and the World Health Organization (WHO)-Monitoring trends and determinants in cardiovascular diseases (MONICA).

Results

In the post-ban period, AMI incidence and mortality rates significantly decreased (relative risk [RR] = 0.89; 95% confidence interval [CI] = 0.81–0.97 and RR = 0.82; 95% CI = 0.71–0.94, respectively). Incidence and mortality rates decreased in both sexes, especially in women, and in people aged 65–74 years. Former and non-smokers (passive smokers) showed diminished incidence rates. Implementation of the ban was not associated with AMI case-fatality. Models tended to be more significant with the WHO-MONICA than with the AHA/ESC definition.

Conclusions

The 2006 Spanish partial smoke-free legislation was associated with a decrease in population AMI incidence and mortality, particularly in women, in people aged 65–74 years, and in passive smokers. These results clarify the association between AMI mortality and the enactment of a partial smoke-free legislation and reinforce the effectiveness of smoking regulations in preventing CHD.     

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)₈-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.