Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins.

Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. SIGNIFICANCE: Enterococcal infections, especially bacteremia and endocarditis, are challenging to treat because E. faecium have acquired resistance to multiple classes of antimicrobials, including ampicillin, aminoglycosides, and glycopeptides. Thus, research on different modes of enterococcal pathogenicity is warranted. This study utilized a proteomic approach to identify MV-associated proteins of different nosocomial E. faecium strains representing four clinically relevant sequence types (STs), namely ST17, ST18, ST78, and ST192. The presented data suggest that E. faecium MVs are involved in virulence and antimicrobial resistance.

Staphylococcus aureus: determinants of human carriage.

Staphylococcus aureus is a common human commensal but carriage varies between e.g. geographic location, age, gender, ethnicity and body niche. The nares, throat and perineum are the most prevalent sites for carriage in the general adult population. Other sites of the skin and the intestine are also frequently colonised. Thus, a successful establishment is dependent on multiple factors. This review describes results from observational studies of S. aureus carriage and the influence bacterial, host and environmental/modifiable factors might have on the relationship.

Staphylococcus aureus nasal carriage is associated with serum 25-hydroxyvitamin D levels, gender and smoking status. The Tromsø Staph and Skin Study.

Vitamin D induces the expression of antimicrobial peptides with activity against Staphylococcus aureus. Thus, we studied the association between serum 25-hydroxyvitamin D (25(OH)D) and S. aureus nasal colonization and carriage. Nasal swabs, blood samples and clinical data from 2,115 women and 1,674 men, aged 30-87 years, were collected in the Tromsø Staph and Skin Study 2007-08, as part of the population-based sixth Tromsø Study. Multivariate logistic regression analyses were stratified by recognized risk factors for S. aureus carriage: sex, age and smoking. In non-smoking men, we observed a 6.6% and 6.7% decrease in the probability of S. aureus colonization and carriage, respectively, by each 5 nmol/l increase in serum 25(OH)D concentration (P < 0.001 and P = 0.001), and serum 25(OH)D > 59 nmol/l and ≥75 nmol/l as thresholds for ~30% and ~50% reduction in S. aureus colonization and carriage. In non-smoking men aged 44-60 years, the odds ratio for S. aureus colonization was 0.44 (95% confidence interval, 0.28-0.69) in the top tertile of serum 25(OH)D versus the bottom tertile. In women and smokers there were no such associations. Our study supports that serum vitamin D is a determinant of S. aureus colonization and carriage.

Oncogenic potentials of the human polyomavirus regulatory proteins.

The polyomaviruses BK, JC and SV40 are common in the human population. Their DNA genomes encode large T-antigen, small t-antigen, agnoprotein, and the capsid proteins VP1-3. Studies with these viruses have contributed extensively to the understanding of processes such as replication, transcriptional and posttranscriptional regulation, and cell cycle control. All three viruses can transform human cells in vitro, can induce tumours in animal models, and are strongly association with certain human cancers. It is generally assumed that large T-antigen is the major protein involved in neoplastic processes and that large T-antigen predominantly exerts its effect through deregulation of the tumour suppressors p53 and the retinoblastoma family members. However, additional properties of large T-antigen as well as the other viral proteins contribute to oncogenic processes. This review presents the different mechanisms by which the polyomavirus proteins can induce transformation and discusses which mechanisms may be operational in polyomavirus-positive cancers.

Competition affects gene flow from oilseed rape (female symbol) to Brassica rapa (male symbol).

Unlike most studies on hybridisation between oilseed rape and Brassica rapa, this study focused on hybridisation with oilseed rape as the maternal parent. This is a key cross because, assuming that plastids are inherited maternally, F(1)-hybrid production with maternal oilseed rape (B. napus) is the only transgene escape route from transplastomic oilseed rape. We investigated such F(1)-hybrid production in winter oilseed rape co-cultivated with weedy B. rapa at three plant densities each with two proportions of the different species. The paternity of the progeny produced on oilseed rape was assessed, and several fitness parameters were determined in oilseed rape mother plants in order to correlate hybridisation and plant competition. At higher density, the vegetative fitness per mother plant decreased significantly, but the density only affected the frequency of F(1)-hybrids significantly (a decrease) in the treatment with equal proportions of each species. As to the proportions, at higher B. napus frequencies, there were fewer F(1)-hybrids per mother plant and a significant increase in most biomass components. Thus, B. rapa was the stronger competitor in its effect on both the vegetative and reproductive fitness in B. napus, and the hybridisation frequency. In conclusion, the relative frequency of the two species was a more influential parameter than the density. Hybridisation with B. napus as the female will be most likely at current field densities of B. napus and when B. rapa is an abundant weed.

The multifunctional roles of the four-and-a-half-LIM only protein FHL2.

Numerous cellular processes require the concerted action of multiple proteins that assemble in functional complexes. Protein-protein interaction domains allow specific proteins to combine with certain partners. Specificity of protein-protein association can be obtained by an interaction code predicted by conserved amino acid sequences. One of the protein-protein interaction motifs is the LIM domain, a conserved cysteine-rich module present in more than 100 different human proteins. The human four-and-a-half-LIM-only protein family consists of the members FHL1, FHL2, FHL3, FHL4 and ACT. They are expressed in a cell- and tissue-specific manner and participate in various cellular processes, including regulation of cell survival, transcription and signal transduction. Here, we review the current knowledge of the best-studied member of this family, FHL2. We describe the transcription regulation, the expression profile, the interaction partners, the subcellular localization, the biological functions and discuss the possible involvement of FHL2 in human diseases.

Effect of recombinant factor VIIa (Novoseven) on thrombocytopenia-like conditions in vitro.

Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Copenhagen, Denmark) may help to promote hemostasis in patients with thrombocytopenia. We used two in vitro models of thrombin generation to evaluate this effect. The reconstituted model contained tissue factor (TF)-expressing monocytes, unactivated platelets, isolated plasma coagulation proteins, and calcium. Platelet activation and thrombin generation were measured in timed aliquots. In the plasma-based model, thrombin generation was measured continuously after the addition of lipidated TF and calcium to platelet-rich plasma using a slowly cleaved fluorescent substrate. Thrombocytopenic conditions were mimicked by decreasing the platelet density. In both systems, a platelet density-dependent lowering of the thrombin-generation peak was observed. Addition of rFVIIa to samples with low platelet density (6700 to 10000/microL) increased the initial thrombin generation in both systems without normalizing thrombin-generation curves. The magnitude of the rFVIIa effect was most pronounced in the plasma-based model. Platelet activation was not significantly delayed at low platelet density in the reconstituted model. Addition of rFVIIa to samples with low platelet density caused faster platelet activation, most likely as a consequence of the increased initial thrombin generation. The data suggest that rFVIIa may help to achieve hemostasis at low platelet densities by increasing the initial thrombin generation, thereby compensating for low platelet number.

Pharmacokinetics and safety of FFR-rFVIIa after single doses in healthy subjects.

FFR-rFVIIa is an antithrombotic agent, which has also proven to have antirestenotic properties in animal models. FFR-rFVIIa is a modified recombinant FVIIa in which the catalytic site is irreversibly inactivated by a synthetic tripeptide covalently bound with the FVIIa molecule. The modified rFVIIa retains its tissue factor (TF) binding capacity but is otherwise enzymatically inactive. A double-blind, placebo-controlled, randomized dose escalation trial was conducted to investigate eight single i.v. doses of FFR-rFVIIa (0.01, 0.02, 0.05, 0.08, 0.12, 0.18, 0.27, or 0.40 mg/kg body weight) in healthy male volunteers (n = 62). Safety, pharmacokinetics, and pharmacodynamics of FFR-rFVIIa were assessed. Mean (SD)AUC0-infinity ranged from 0.35 (0.11) to 28.8 (3.5)microg.h/ml, and mean Cmax ranged from 0.078 (0.019) to 4.8 (0.7) microg/ml. The mean elimination half-life ranged from 3.8 to 5.8 hours. Mean AUC0-infinity increased with increasing dose levels. Cmax appeared to be proportional to the dose level, with the exception of the lowest dose level. A dose-dependent prolongation of the prothrombin time was found, demonstrating that FFR-rFVIIa inhibited coagulation via the TF-dependent pathway FFR-rFVIIa was generally well tolerated at all dose levels studied.

Comparison of the factor VII:C clot analysis and a modified activated factor VII analysis for monitoring factor VII activity in patients treated with recombinant activated factor VII (NovoSeven).

Bleeding episodes in haemophilia A and B inhibitor patients are now frequently treated with recombinant activated factor VII (NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark). Until now, the FVII:C coagulation assay has been used to monitor NovoSeven-mediated coagulation. However, a new assay (Staclot VIIa-rTF, Diagnostica Stago, France) has been designed to specifically detect activated factor (F)VII. Replacement of the buffer supplied by the manufacturer with a PIPES buffer containing BSA (modified FVIIa assay), resulted in a linear standard curve, greater sample stability and a reduced coefficient of variation. The FVII:C assay and the modified FVIIa assay were compared in a recovery experiment using the International FVIIa standard No 89/688(IS). Recovery of FVIIa was 93-97% for the modified FVIIa assay and 91-115% for the FVII:C assay. However, because samples in the FVII:C assay were not parallel to the standard curve, confidence limits for recovery were as wide as 67-130% compared with 92-106% for the FVIIa assay. In conclusion, a modified version of the Staclot VIIa-rTF assay, suitable for monitoring treatment with NovoSeven, even at low concentrations, has been developed. It provides an alternative to the FVII:C assay, which is not suitable for monitoring FVIIa at low concentrations.

Decline of factor VIII and factor IX inhibitors during long-term treatment with NovoSeven.

Recombinant factor VIIa (rFVIIa) (NovoSeveng) is used to treat bleeding episodes in hemophilia A and B patients with inhibitor antibodies against factor VIII (FVIII) and factor IX. rFVIIIa has been studied in home treatment of mild-to-moderate joint, muscle, and mucocutaneous bleeds to assess safety and efficacy. Treatment with other factor concentrates was allowed according to treating physician's judgment. Blood samples were drawn before study start and after 6 and 12 months. It has thus been possible to follow the inhibitor titres during this period. Analyses of 53 patients (49 hemophilia A, four hemophilia B) showed inhibitor levels up to 1,208 BU/ml before study start. Based on the first analysis, hemophilia A patients were divided into high responders (> 5 BU/ml; 28 patients), low responders (> 1 and < 5 BU/ml; 15 patients) and very low responders (< or = 1 BU/ml; six patients). In high responders receiving rFVIIa as only treatment, FVIII inhibitor titre decreased to one-third of the initial level. For high responders receiving other factor treatments such as FVIII or prothrombin complex concentrates, inhibitor titre remained unchanged. Titres for low responders and very low responders remained unchanged independent of treatment. Thus, when rFVIIa is used as the only coagulation factor to treat hemophilia A/B high-responder inhibitor patients, inhibitor level declines significantly.

Providing a sound habitat for man in space.

Microbiological growth on materials in an indoor environment contributes to the well known "sick building syndrome". The inhabitants' health and well-being is affected by injurious vapours and odours given off to the air. This is particularly pronounced in new and better tightened houses with unconventional building materials and wider employment of air conditioning. The European Space Agency has recognized the problems to be expected in a totally closed and self-supported long-term habitat and has induced work on the selection of materials, resistant to microbiological growth, and on other microbial contamination control measures. Requirements and procedures are being established as a basis for the microbiological cleanliness of the manned space environment and for the avoidance of microbiological growth on materials and equipment. Methods are being developed, suitable for testing and predicting the resistivity to microbiological growth of materials to be used in long-term space habitats.

Quenching of the amidolytic activity of one-chain tissue-type plasminogen activator by mutation of lysine-416.

In contrast to most other serine proteases, tissue-type plasminogen activator (t-PA) possesses enzymatic activity as the one-chain zymogen form. The hypothesis that lysine residues 277 or 416 may be involved in stabilization of an active conformation of one-chain t-PA via salt-bridge formation with aspartic acid residue 477 was tested by site-directed mutagenesis. Four recombinant t-PA mutants were constructed. The amidolytic activities of these analogues were compared to that of authentic t-PA. Substitution of arginine-275 provided an analogue [( R275G]t-PA) resistant to plasmin cleavage. The amidolytic activity of [R275G]t-PA was comparable to that of authentic one-chain t-PA, and so was the activity of [R275L,K277L]t-PA, in which additional substitution of lysine residue 277 was carried out. This suggested that its presence was nonessential for obtaining one-chain t-PA activity. In contrast, substitution of lysine residue 416 to obtain [K416S]t-PA and [K416S,H417T]t-PA resulted in substantial quenching of amidolytic one-chain activity. As expected, the amidolytic activities of the two-chain forms were less affected by the substitution. Involvement of lysine residue 416 in one-chain t-PA activity was also indicated by decreased activities of [K416S]t-PA and [K416S,H417T]t-PA with plasminogen as the substrate. The one-chain activity of the lysine residue 416 substitution analogues was partially restored in the presence of fibrin. This could indicate that strong ligands such as fibrin might provide an alternative stabilization of the active conformation of one-chain t-PA.

Fibrin affinity and clearance of t-PA deletion and substitution analogues.

To investigate structure-function relationships in tissue-type plasminogen activator (t-PA) we deleted the following domains in the heavy chain: a) The epidermal growth factor domain (t-PA del. G), b) the finger domain, and the epidermal growth factor domain (t-PA del. FG), and c) the finger, the epidermal growth factor and Kringle 1 (t-PA del. FGK1). To study specifically the function of the growth factor domain we made two substitutions of d) 8 amino acids (consensus sequence) in the growth factor domain (t-PA G-CS) and e) the whole domain with factor IX growth factor domain (t-PA G-IX). Finally, f) an analogue with substitution in the finger domain (fibronectin consensus sequence) was constructed (t-PA F-CS). A reduced fibrin binding of all the analogues was found. The fibrin stimulated activity of all analogues was also reduced and correlated to the fibrin binding. In contrast, the activity of the analogues in the clot lysis assay and the plate assay were only slightly reduced as compared to authentic t-PA. This suggested that at high fibrin concentrations the decreased fibrin affinity was less critical for obtaining a high fibrinolytic activity. All analogues had a prolonged half-life in vivo as compared to authentic t-PA. The assumption of clearance mechanism involving mainly the growth factor region (or Kringle 1) was not challenged by the observation of a prolonged half-life for the substitution analogue t-PA F-CS.2+off

The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage.

A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.

The role of microorganisms in the formation of pitch deposits in pulp and paper mills.

The cause of pitch deposit formation seems still not fully understood. The work reported here demonstrates that microorganisms effect the agglomeration of emulgated resin droplets and the formation of sticky precipitates. Pitch deposits from mills consist mainly of ethanol-soluble resins. It is also the ethanol-soluble fraction of wood resins which forms stable emulsions and which is easily agglomerated by microorganisms. Pitch deposits, collected from various pulp and paper mills, were all found to contain large amounts of microorganisms. Sterile resin emulsions prepared from pitch deposits remained stable over long periods. After inoculation with microorganisms the emulsions were destabilized and the resins completely precipitated as sticky lumps. Various bacteria and fungi are capable of agglomerating the resins, but species isolated from water, pulp and slime in paper mills were usually most effective. Resins from fresh wood were precipitated at a faster rate than aged resins. Problems of pitch formation can be considerably reduced when microbial growth is kept under control in the production system. To be effective, the control measures, e.g. slimicides, must be applied at the right place and time, and in the correct concentrations. This presupposes a thorough knowledge of the plant's microbiological condition which can only be obtained by microbiological examination. Practical cases of the appropriate application of biocides in pulp and paper mill systems are discussed.