A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Influence of nanofibers on growth and gene expression of human tendon derived fibroblast

Background  

Rotator cuff tears are a common and frequent lesion especially in older patients. The mechanisms of tendon repair are not fully understood. Common therapy options for tendon repair include mini-open or arthroscopic surgery. The use of growth factors in experimental studies is mentioned in the literature. Nanofiber scaffolds, which provide several criteria for the healing process, might be a suitable therapy option for operative treatment. The aim of this study was to explore the effects of nanofiber scaffolds on human tendon derived fibroblasts (TDF's), as well as the gene expression and matrix deposition of these fibroblasts.     

Capsular Polysaccharide Production in Enterococcus faecalis and Contribution of CpsF to Capsule Serospecificity

Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.Enterococcus faecalis is a gram-positive bacterium commonly found as a commensal organism in the gastrointestinal tracts of most mammals. E. faecalis is one of the leading causes of hospital-acquired urinary tract infections, bacteremia, and surgical-site infections (29). The development of multiple antibiotic resistances, including resistance to vancomycin, makes treatment of enterococcal infections difficult (11). The 2004 National Nosocomial Infections Surveillance report indicated that nearly 30% of enterococci isolated from clinical settings were resistant to vancomycin, constituting a 12% rise from the previous 5 years (26). The development of alternative therapies to treat enterococcal infections has frequently been suggested due to rising percentages of antibiotic-resistant enterococcal strains (13-15, 19).Capsular polysaccharides are major contributors to the virulence of many microorganisms. The presence of capsule allows these microbes to escape detection and clearance by the host immune system (9, 27, 30, 41). There have been several publications regarding the role of cell wall polysaccharides in the pathogenesis of enterococcal infections (10, 13, 17, 37, 43). Several attempts have been made to establish a serotyping system for E. faecalis capsular polysaccharides (16, 23, 35, 36). These serotyping schemes include differences in capsular polysaccharide antigens but are also based on differences in surface antigens, including lipoteichoic acid (16, 38). To date, only one study has linked genetic evidence with capsule production (12). Two loci that have been reported to contain putative genes for capsule production are the epa and cps operons (10, 42). The polysaccharide produced by the epa locus is thought to be the cell wall rhamnopolymer (10), but it cannot be detected on the surface of the bacterium (43). Although rhamnopolymer production is reported to be abrogated by mutation (43), the full nature of rhamnopolymer production is yet to be determined for many E. faecalis strains. Probing the genomes of serotype A and B strains with a probe specific to the cps locus, including the genes cpsA and cpsB, identified a single ClaI restriction fragment for serotypes A and B (16). However, multiple ClaI restriction fragments were identified in serotypes C and D (16), suggesting that the genes responsible for capsule production in serotypes C and D were absent in serotypes A and B. Furthermore, the hybridization pattern between serotype C and D strains indicated a single restriction fragment polymorphism, but the basis on which genes were different between the two serotypes was not fully characterized (16). Studies based on the serotyping scheme proposed by Hufnagel et al. (17) have shown that serotype C and D strains are much more resistant to opsonophagoctyosis by neutrophils in the presence of normal human serum. More recently, a study by McBride et al. indicated that serotype C clinical isolates harbored a greater repertoire of antibiotic resistance cassettes and were more likely to possess multiple virulence factors than the other serotypes, suggesting that the presence of the capsule is associated with pathogenic lineages of E. faecalis (17, 24).It is essential to understand the underlying mechanisms of capsule production in E. faecalis because of ongoing efforts to develop alternative therapies targeting capsule. Here, we used a novel vector system for creating isogenic, in-frame deletion mutants to analyze the genetic basis for capsule production and serotype specificity. Our results show that only serotype C and D strains of E. faecalis produce capsular polysaccharides, based on the observation that deletions of cpsC, cpsD, cpsE cpsG, and cpsI abolish the production of capsule. In conjunction with these observations, we also demonstrated that the presence of capsule prevents detection of lipoteichoic acid on the surface of serotype C and D strains but not on unencapsulated strains. Our data also show that CpsF is responsible for the difference in serospecificity between serotype C and D strains.     

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.     

Periodontal therapy increases neutrophil extracellular trap degradation

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.     

Multidimensional prognostic indices for use in COPD patient care. A systematic review

Background

A growing number of prognostic indices for chronic obstructive pulmonary disease (COPD) is developed for clinical use. Our aim is to identify, summarize and compare all published prognostic COPD indices, and to discuss their performance, usefulness and implementation in daily practice.

Methods

We performed a systematic literature search in both Pubmed and Embase up to September 2010. Selection criteria included primary publications of indices developed for stable COPD patients, that predict future outcome by a multidimensional scoring system, developed for and validated with COPD patients only. Two reviewers independently assessed the index quality using a structured screening form for systematically scoring prognostic studies.

Results

Of 7,028 articles screened, 13 studies comprising 15 indices were included. Only 1 index had been explored for its application in daily practice. We observed 21 different predictors and 7 prognostic outcomes, the latter reflecting mortality, hospitalization and exacerbation. Consistent strong predictors were FEV₁ percentage predicted, age and dyspnoea. The quality of the studies underlying the indices varied between fairly poor and good. Statistical methods to assess the predictive abilities of the indices were heterogenic. They generally revealed moderate to good discrimination, when measured. Limitations: We focused on prognostic indices for stable disease only and, inevitably, quality judgment was prone to subjectivity.

Conclusions

We identified 15 prognostic COPD indices. Although the prognostic performance of some of the indices has been validated, they all lack sufficient evidence for implementation. Whether or not the use of prognostic indices improves COPD disease management or patients'' health is currently unknown; impact studies are required to establish this.