Genome analysis of DNA repair genes in the alpha proteobacterium <Emphasis Type="Italic">Caulobacter crescentus</Emphasis>

Background  

The integrity of DNA molecules is fundamental for maintaining life. The DNA repair proteins protect organisms against genetic damage, by removal of DNA lesions or helping to tolerate them. DNA repair genes are best known from the gamma-proteobacterium Escherichia coli, which is the most understood bacterial model. However, genome sequencing raises questions regarding uniformity and ubiquity of these DNA repair genes and pathways, reinforcing the need for identifying genes and proteins, which may respond to DNA damage in other bacteria.     

Apoptosis and p53 expression in rat adjuvant arthritis

The kinetics of apoptosis and the apoptosis-regulating gene p53 in adjuvant arthritis (AA) were investigated to assess the value of the AA rat model for testing apoptosis-inducing therapies. Very few terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-positive cells were detected during the early phases of AA, but on day 23 (chronic arthritis) the percentage of TUNEL-positive cells was significantly increased. Expression of p53 in synovial tissue gradually increased from days 5-23, which was markedly higher than p53 levels in rheumatoid arthritis (RA) synovium. Significant apoptosis only occurs late in rat AA and is concordant with marked p53 overexpression, making it useful model for testing proapoptotic therapies, but rat AA is not the best model for p53 gene therapy because dramatic p53 overexpression occurs in the latter stages of the disease.     

Identification of a haemolysin-like peptide with antibacterial activity using the draft genome sequence of Staphylococcus epidermidis strain A487

Our interest in Staphylococcus epidermidis strain A487 was prompted by the unusual nature of its inhibitory activity in screening tests against methicillin-resistant Staphylococcus aureus isolates. The inhibitory activity was detected in deferred antagonism tests only if the agar plate was preheated for at least 35 min at ≥ 55 °C before inoculation of the indicator bacteria, this phenomenon indicating possible involvement of a heat-labile immunity agent or protease. The inhibitor was purified to homogeneity by ammonium sulphate precipitation, followed by cation-exchange and reversed-phase chromatography. Tandem MS revealed a novel peptide of molecular weight 2588.4 Da. The draft genome sequence of strain A487 was determined using 454 GS FLX technology, allowing the identification of the structural gene (hlp) encoding the mature peptide MQFITDLIKKAVDFFKGLFGNK. The deduced amino acid sequence of peptide 487 exhibited 70.8% similarity to that of a putative haemolysin from Staphylococcus cohnii. Analysis of the genome of strain A487 showed several additional inhibitor-encoding genes, including hld, the determinant for staphylococcal δ-lysin. This work indicates that potentially useful inhibitors could be overlooked in agar-based inhibitor screening programmes lacking a heat pretreatment step and also highlights the utility of draft genome sequence examination in antibacterial agent discovery.     

(Cost)-effectiveness of a multi-component intervention for adults with epilepsy: study protocol of a Dutch randomized controlled trial (ZMILE study)

Background

In patients with epilepsy, poor adherence to anti-epileptic drugs has been shown to be the most important cause of poorly controlled epilepsy. Furthermore, it has been noted that the quality of life among patients with epilepsy can be improved by counseling and treatments aimed at increasing their self-efficacy and concordance, thus stimulating self-management skills. However, there is a need for evidence on the effectiveness of such programs, especially within epilepsy care. Therefore, we have developed a multi-component intervention (MCI) which combines a self-management/education program with e-Health interventions. Accordingly, the overall objective of this study is to assess the (cost)-effectiveness and feasibility of the MCI, aiming to improve self-efficacy and concordance in patients with epilepsy.

Methods

A RCT in two parallel groups will be conducted to compare the MCI with a control condition in epilepsy patients. One hundred eligible epilepsy patients will be recruited and allocated to either the intervention or control group. The intervention group will receive the MCI consisting of a self-management/education program of six meetings, including e-Health interventions, and will be followed for 12 months. The control group will receive care as usual and will be followed for 6 months, after which patients will be offered the possibility of participating in the MCI. The study will consist of three parts: 1) a clinical effectiveness study, 2) a cost-effectiveness study, and 3) process evaluation. The primary outcome will be self-efficacy. Secondary outcomes include adherence, side effects, change in seizure severity & frequency, improved quality of life, proactive coping, and societal costs. Outcome assessments will be done using questionnaires at baseline and after 3, 6, 9, and 12 months (last two applicable only for intervention group).

Discussion

In times of budget constraints, MCI could be a valuable addition to the current healthcare provision for epilepsy, as it is expected that higher concordance and self-efficacy will result in reduced use of healthcare resources and an increased QOL. Accordingly, this study is aimed helping patients to be their own provider of health care, shifting epilepsy management from professionals to self-care by patients equipped with appropriate skills and tools.

Trial registration number

NTR4484.

     

Promising potential of new generation translocator protein tracers providing enhanced contrast of arthritis imaging by positron emission tomography in a rat model of arthritis

Introduction

The role of popliteal cysts and subgastrocnemius bursitis in knee joint homeostasis is uncertain. The aim of this study is to describe cross-sectional associations between popliteal cysts, subgastrocnemius bursitis, knee symptoms and structural abnormalities in older adults.

Methods

A cross-sectional sample of 900 randomly-selected subjects (mean age 63 years, 48% female) were studied. Knee pain, stiffness and dysfunction were assessed by self-administered Western Ontario McMaster Osteoarthritis Index (WOMAC) questionnaire. Radiographic knee osteophyte and joint space narrowing (JSN) were recorded. Magnetic resonance imaging (MRI) was utilized to assess popliteal cysts, subgastrocnemius bursitis, cartilage defects and bone marrow lesions (BMLs).

Results

Popliteal cysts were present in 11.7% and subgastrocnemius bursitis in 12.7% of subjects. Subgastrocnemius bursitis was more common in those with popliteal cyst (36.2% versus 9.7%, P <0.01). In multivariable analyses, popliteal cysts were significantly associated with increased osteophytes in both medial and lateral tibiofemoral compartments while subgastrocnemius bursitis was associated with increased osteophytes and JSN in the medial tibiofemoral compartment. Both were significantly associated with cartilage defects in all compartments, and with BMLs in the medial tibiofemoral compartment. Furthermore, both popliteal cysts and subgastrocnemius bursitis were significantly associated with increased weight-bearing knee pain but these associations became non-significant after adjustment for cartilage defects and BMLs.

Conclusions

Popliteal cysts and subgastrocnemius bursitis are associated with increased symptoms as well as radiographic and MRI-detected joint structural abnormalities. Longitudinal data will help resolve if they are a consequence or a cause of knee joint abnormalities.     

Nordic Walking improves daily physical activities in COPD: a randomised controlled trial

Background

In patients with COPD progressive dyspnoea leads to a sedentary lifestyle. To date, no studies exist investigating the effects of Nordic Walking in patients with COPD. Therefore, the aim was to determine the feasibility of Nordic Walking in COPD patients at different disease stages. Furthermore we aimed to determine the short- and long-term effects of Nordic Walking on COPD patients'' daily physical activity pattern as well as on patients exercise capacity.

Methods

Sixty COPD patients were randomised to either Nordic Walking or to a control group. Patients of the Nordic Walking group (n = 30; age: 62 ± 9 years; FEV₁: 48 ± 19% predicted) underwent a three-month outdoor Nordic Walking exercise program consisting of one hour walking at 75% of their initial maximum heart rate three times per week, whereas controls had no exercise intervention. Primary endpoint: daily physical activities (measured by a validated tri-axial accelerometer); secondary endpoint: functional exercise capacity (measured by the six-minute walking distance; 6MWD). Assessment time points in both groups: baseline, after three, six and nine months.

Results

After three month training period, in the Nordic Walking group time spent walking and standing as well as intensity of walking increased (Δ walking time: +14.9 ± 1.9 min/day; Δ standing time: +129 ± 26 min/day; Δ movement intensity: +0.40 ± 0.14 m/s²) while time spent sitting decreased (Δ sitting time: -128 ± 15 min/day) compared to baseline (all: p < 0.01) as well as compared to controls (all: p < 0.01). Furthermore, 6MWD significantly increased compared to baseline (Δ 6MWD: +79 ± 28 meters) as well as compared to controls (both: p < 0.01). These significant improvements were sustained six and nine months after baseline. In contrast, controls showed unchanged daily physical activities and 6MWD compared to baseline for all time points.

Conclusions

Nordic Walking is a feasible, simple and effective physical training modality in COPD. In addition, Nordic Walking has proven to positively impact the daily physical activity pattern of COPD patients under short- and long-term observation.

Clinical trial registration

Nordic Walking improves daily physical activities in COPD: a randomised controlled trial - ISRCTN31525632     

Phylogenetic relationships in genus <Emphasis Type="Italic">Arachis</Emphasis> based on ITS and 5.8S rDNA sequences

Background  

The genus Arachis comprises 80 species and it is subdivided into nine taxonomic sections (Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides, and Triseminatae). This genus is naturally confined to South America and most of its species are native to Brazil. In order to provide a better understanding of the evolution of the genus, we reconstructed the phylogeny of 45 species using the variation observed on nucleotide sequences in internal transcribed spacer regions (ITS1 and ITS2) and 5.8 S of nuclear ribosomal DNA.     

New clinically relevant,orthotopic mouse models of human chondrosarcoma with spontaneous metastasis

Background  

Chondrosarcoma responds poorly to adjuvant therapy and new, clinically relevant animal models are required to test targeted therapy.     

Production of the Bsa Lantibiotic by Community-Acquired Staphylococcus aureus Strains

Lantibiotics are antimicrobial peptides that have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications. Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, some lantibiotics are produced by pathogens and, rather than contributing to food safety and/or health, add to the virulence potential of the producing strains. Indeed, genome sequencing has revealed the presence of genes apparently encoding a lantibiotic, designated Bsa (bacteriocin of Staphylococcus aureus), among clinical isolates of S. aureus and those associated with community-acquired methicillin-resistant S. aureus (MRSA) infections in particular. Here, we establish for the first time, through a combination of reverse genetics, mass spectrometry, and mutagenesis, that these genes encode a functional lantibiotic. We also reveal that Bsa is identical to the previously identified bacteriocin staphylococcin Au-26, produced by an S. aureus strain of vaginal origin. Our examination of MRSA isolates that produce the Panton-Valentine leukocidin demonstrates that many community-acquired S. aureus strains, and representatives of ST8 and ST80 in particular, are producers of Bsa. While possession of Bsa immunity genes does not significantly enhance resistance to the related lantibiotic gallidermin, the broad antimicrobial spectrum of Bsa strongly indicates that production of this bacteriocin confers a competitive ecological advantage on community-acquired S. aureus.Staphylococcus aureus can be a human commensal bacterium, colonizing the skin and mucosal surfaces such as the nares, pharynx, and vagina in approximately 25 to 40% of the population. However, it is also a human pathogen that can cause epidemics of invasive disease. Genome sequencing of S. aureus strains has highlighted that the species is highly clonal, with approximately 78% of the genes being conserved and representing the core genome. The remaining 22% of the genes, which are variable and include those present on genomic islands, pathogenicity islands, prophages, integrated plasmids, and transposons, can in turn be regarded as an accessory genome (for a review, see reference 19) that provides a means via which S. aureus can evolve to adapt to particular niches and environmental pressures. The environmental pressure that has most strongly influenced S. aureus evolution in the past century has been the development and application of different antibiotics. These advancements have dictated that the strains that have flourished in hospitals, most notably hospital-acquired methicillin-resistant S. aureus (HA-MRSA) strains, tend to be multidrug resistant but suffer from a concomitant reduction in fitness relative to isolates from the community, due to being encumbered with staphylococcal cassette chromosome mec (SCCmec) types I to III and additional antibiotic resistance genes (48, 55). The negative consequences of this reduction in fitness are, however, mitigated by the reduction in competition from the human commensal microbiota by antibiotic exposure.Since the late 1990s, MRSA infections have been detected among the general population and among healthy individuals (typically children and young adults) who lack traditional risk factors (26). It was apparent that the S. aureus strains responsible for these community-acquired MRSA (CA-MRSA) infections were genetically distinct from their HA counterparts, possessing the more simple type IV (and to a lesser extent, type V and VII) allelic versions of SCCmec (13, 55) and fewer antibiotic resistance genes (20). While this fact indicated that these strains might represent less of a health care challenge than the HA strains, it quickly became apparent that the enhanced competitiveness of these strains, resulting in rapid growth (CA-MRSA strains grow much faster than HA-MRSA strains) (4) and increased virulence (67) of CA-MRSA, meant that any delay in switching from the β-lactam antibiotics normally used to treat infections of unknown etiology could have very serious medical implications, including death. Indeed, paradoxically, CA-MRSA strains have since spread to hospitals and have been responsible for a number of infections.In contrast to HA-MRSA strains, which by virtue of their multidrug-resistant nature, coupled with exposure to antibiotics, have a selective advantage over other microorganisms in the hospital environment, CA-MRSA strains, like commensal S. aureus strains, often face stiff competition from the natural flora of healthy individuals. It has been speculated that the production of an antimicrobial compound may provide CA-MRSA isolates with a competitive advantage in such environments (4, 14). The theory was first suggested when sequencing of strain FPR3757 (part of the virulent USA300 clonal group) revealed the presence of bsa (bacteriocin of S. aureus) genes, which resembled those associated with production of the epidermin subgroup of lantibiotics (2, 60). Lantibiotics are ribosomally produced, posttranslationally modified peptide antibiotics that are generally active against bacterial species which are closely related to the producing organism, and these antimicrobials are thought to have a role in niche competition in many natural environments (41). Lantibiotics have been the focus of much attention in recent years with a view to clinical, veterinary, and food applications (10, 72). Although many lantibiotics are produced by food-grade bacteria or bacteria generally regarded as safe, there have also been a few examples of lantibiotic production by pathogens (11, 46, 69). In this instance, despite the identification of the bsa genes, the production of a lantibiotic by CA-MRSA isolates has remained speculative. Indeed, to date, there has been only one confirmed example of a lantibiotic, i.e., staphylococcin C55 (46), produced by S. aureus and no definitive evidence that CA- (or HA)-MRSA strains produce such compounds. There is, however, some evidence to suggest that staphylococcin Au-26, which is produced by a vaginal isolate of S. aureus and has an inhibitory spectrum encompassing lactobacilli isolated from the endocervix and representative strains of Staphylococcus hominis, Staphylococcus warneri, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus mutans, Lactococcus spp., and oral Neisseria spp., may also be a lantibiotic (63). Here, 17 years after its initial characterization, we have carried out a closer inspection of staphylococcin Au-26 and the associated producer and have established that the staphylococcin Au-26 and Bsa genetic loci are almost identical. Prompted by this finding, we employed a combination of mutagenesis and mass spectrometry (MS) to reveal that these genes are functional in a number of other staphylococci, including a large percentage of CA-MRSA isolates. We suggest that, as a consequence of eliminating competing human microbiota, this lantibiotic contributes strongly to the fitness of these community-associated isolates.