The complete genome sequence and analysis of Corynebacterium diphtheriae NCTC13129

Corynebacterium diphtheriae is a Gram-positive, non-spore forming, non-motile, pleomorphic rod belonging to the genus Corynebacterium and the actinomycete group of organisms. The organism produces a potent bacteriophage-encoded protein exotoxin, diphtheria toxin (DT), which causes the symptoms of diphtheria. This potentially fatal infectious disease is controlled in many developed countries by an effective immunisation programme. However, the disease has made a dramatic return in recent years, in particular within the Eastern European region. The largest, and still on-going, outbreak since the advent of mass immunisation started within Russia and the newly independent states of the former Soviet Union in the 1990s. We have sequenced the genome of a UK clinical isolate (biotype gravis strain NCTC13129), representative of the clone responsible for this outbreak. The genome consists of a single circular chromosome of 2 488 635 bp, with no plasmids. It provides evidence that recent acquisition of pathogenicity factors goes beyond the toxin itself, and includes iron-uptake systems, adhesins and fimbrial proteins. This is in contrast to Corynebacterium’s nearest sequenced pathogenic relative, Mycobacterium tuberculosis, where there is little evidence of recent horizontal DNA acquisition. The genome itself shows an unusually extreme large-scale compositional bias, being noticeably higher in G+C near the origin than at the terminus.     

The use of ultrasonic imaging to evaluate the effect of protozoan grazing and movement on the topography of bacterial biofilms

AIMS: This study evaluated the effect of protozoan movement and grazing on the topography of a dual-bacterial biofilm using both conventional light microscopy and a new ultrasonic technique. METHODS AND RESULTS: Coupons of dialysis membrane were incubated in Chalkley's medium for 3 days at 23 degrees C in the presence of bacteria (Pseudomonas aeruginosa and Klebsiella aerogenes) alone, or in co-culture with the flagellate Bodo designis, the ciliate Tetrahymena pyriformis or the amoeba Acanthamoeba castellanii. Amoebic presence resulted in a confluent biofilm similar to the bacteria-only biofilm while the flagellate and ciliate created more diverse biofilm topographies comprising bacterial microcolonies and cavities. CONCLUSIONS: The four distinct biofilm topographies were successfully discerned with ultrasonic imaging and the method yielded information similar to that obtained with conventional light microscopy. SIGNIFICANCE AND IMPACT OF THE STUDY: Ultrasonic imaging provides a potential way forward in the development of a portable, nondestructive technique for profiling the topography of biofilms in situ, which might aid in the future management of biofouling.     

Statistical considerations of optimal study design for human plasma proteomics and biomarker discovery

A mass spectrometry-based plasma biomarker discovery workflow was developed to facilitate biomarker discovery. Plasma from either healthy volunteers or patients with pancreatic cancer was 8-plex iTRAQ labeled, fractionated by 2-dimensional reversed phase chromatography and subjected to MALDI ToF/ToF mass spectrometry. Data were processed using a q-value based statistical approach to maximize protein quantification and identification. Technical (between duplicate samples) and biological variance (between and within individuals) were calculated and power analysis was thereby enabled. An a priori power analysis was carried out using samples from healthy volunteers to define sample sizes required for robust biomarker identification. The result was subsequently validated with a post hoc power analysis using a real clinical setting involving pancreatic cancer patients. This demonstrated that six samples per group (e.g., pre- vs post-treatment) may provide sufficient statistical power for most proteins with changes>2 fold. A reference standard allowed direct comparison of protein expression changes between multiple experiments. Analysis of patient plasma prior to treatment identified 29 proteins with significant changes within individual patient. Changes in Peroxiredoxin II levels were confirmed by Western blot. This q-value based statistical approach in combination with reference standard samples can be applied with confidence in the design and execution of clinical studies for predictive, prognostic, and/or pharmacodynamic biomarker discovery. The power analysis provides information required prior to study initiation.     

A Dutch guideline for the treatment of scoliosis in neuromuscular disorders

Background

Children with neuromuscular disorders with a progressive muscle weakness such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy frequently develop a progressive scoliosis. A severe scoliosis compromises respiratory function and makes sitting more difficult. Spinal surgery is considered the primary treatment option for correcting severe scoliosis in neuromuscular disorders. Surgery in this population requires a multidisciplinary approach, careful planning, dedicated surgical procedures, and specialized after care.

Methods

The guideline is based on scientific evidence and expert opinions. A multidisciplinary working group representing experts from all relevant specialties performed the research. A literature search was conducted to collect scientific evidence in answer to specific questions posed by the working group. Literature was classified according to the level of evidence.

Results

For most aspects of the treatment scientific evidence is scarce and only low level cohort studies were found. Nevertheless, a high degree of consensus was reached about the management of patients with scoliosis in neuromuscular disorders. This was translated into a set of recommendations, which are now officially accepted as a general guideline in the Netherlands.

Conclusion

In order to optimize the treatment for scoliosis in neuromuscular disorders a Dutch guideline has been composed. This evidence-based, multidisciplinary guideline addresses conservative treatment, the preoperative, perioperative, and postoperative care of scoliosis in neuromuscular disorders.     

Meningococcal genetic variation mechanisms viewed through comparative analysis of serogroup C strain FAM18

The bacterium Neisseria meningitidis is commonly found harmlessly colonising the mucosal surfaces of the human nasopharynx. Occasionally strains can invade host tissues causing septicaemia and meningitis, making the bacterium a major cause of morbidity and mortality in both the developed and developing world. The species is known to be diverse in many ways, as a product of its natural transformability and of a range of recombination and mutation-based systems. Previous work on pathogenic Neisseria has identified several mechanisms for the generation of diversity of surface structures, including phase variation based on slippage-like mechanisms and sequence conversion of expressed genes using information from silent loci. Comparison of the genome sequences of two N. meningitidis strains, serogroup B MC58 and serogroup A Z2491, suggested further mechanisms of variation, including C-terminal exchange in specific genes and enhanced localised recombination and variation related to repeat arrays. We have sequenced the genome of N. meningitidis strain FAM18, a representative of the ST-11/ET-37 complex, providing the first genome sequence for the disease-causing serogroup C meningococci; it has 1,976 predicted genes, of which 60 do not have orthologues in the previously sequenced serogroup A or B strains. Through genome comparison with Z2491 and MC58 we have further characterised specific mechanisms of genetic variation in N. meningitidis, describing specialised loci for generation of cell surface protein variants and measuring the association between noncoding repeat arrays and sequence variation in flanking genes. Here we provide a detailed view of novel genetic diversification mechanisms in N. meningitidis. Our analysis provides evidence for the hypothesis that the noncoding repeat arrays in neisserial genomes (neisserial intergenic mosaic elements) provide a crucial mechanism for the generation of surface antigen variants. Such variation will have an impact on the interaction with the host tissues, and understanding these mechanisms is important to aid our understanding of the intimate and complex relationship between the human nasopharynx and the meningococcus.     

Free and total cortisol levels in semelparous and iteroparous chinook salmon

Total cortisol, free cortisol and percent free cortisol were all significantly higher in semelparous male chinook salmon Oncorhynchus tshawytscha than in iteroparous and immature fish. The findings suggest that the regulation of both total and free cortisol concentrations may play key roles in mediating the post-spawning death of semelparous Pacific salmon.     

The hand of the stacked-disk aggregate of tobacco mosaic virus protein

The hand of the helical arrangement of subunits in the stacked-disk form of tobacco mosaic virus protein has been determined from electron microscopy. This and previous structural results taken together indicate that the near-axial intersubunit contacts between disks are similar to those within a single disk but quite different from those in the virus. Discrepancies between the structures of single disks and stacked disks are attributed to cleavage of the protein within the stacked-disk rod.