Early combination disease-modifying antirheumatic drug therapy and tight disease control improve long-term radiologic outcome in patients with early rheumatoid arthritis: the 11-year results of the Finnish Rheumatoid Arthritis Combination Therapy trial

Introduction  

Early treatment of rheumatoid arthritis (RA) has been shown to retard the development of joint damage for a period of up to 5 years. The aim of this study was to evaluate the radiologic progression beyond that time in patients with early RA initially treated with a combination of three disease-modifying antirheumatic drugs (DMARDs) or a single DMARD.     

Integration Preferences of Wildtype AAV-2 for Consensus Rep-Binding Sites at Numerous Loci in the Human Genome

Adeno-associated virus type 2 (AAV) is known to establish latency by preferential integration in human chromosome 19q13.42. The AAV non-structural protein Rep appears to target a site called AAVS1 by simultaneously binding to Rep-binding sites (RBS) present on the AAV genome and within AAVS1. In the absence of Rep, as is the case with AAV vectors, chromosomal integration is rare and random. For a genome-wide survey of wildtype AAV integration a linker-selection-mediated (LSM)-PCR strategy was designed to retrieve AAV-chromosomal junctions. DNA sequence determination revealed wildtype AAV integration sites scattered over the entire human genome. The bioinformatic analysis of these integration sites compared to those of rep-deficient AAV vectors revealed a highly significant overrepresentation of integration events near to consensus RBS. Integration hotspots included AAVS1 with 10% of total events. Novel hotspots near consensus RBS were identified on chromosome 5p13.3 denoted AAVS2 and on chromsome 3p24.3 denoted AAVS3. AAVS2 displayed seven independent junctions clustered within only 14 bp of a consensus RBS which proved to bind Rep in vitro similar to the RBS in AAVS3. Expression of Rep in the presence of rep-deficient AAV vectors shifted targeting preferences from random integration back to the neighbourhood of consensus RBS at hotspots and numerous additional sites in the human genome. In summary, targeted AAV integration is not as specific for AAVS1 as previously assumed. Rather, Rep targets AAV to integrate into open chromatin regions in the reach of various, consensus RBS homologues in the human genome.     

Identification of flowering genes in strawberry, a perennial SD plant

Background  

We are studying the regulation of flowering in perennial plants by using diploid wild strawberry (Fragaria vesca L.) as a model. Wild strawberry is a facultative short-day plant with an obligatory short-day requirement at temperatures above 15°C. At lower temperatures, however, flowering induction occurs irrespective of photoperiod. In addition to short-day genotypes, everbearing forms of wild strawberry are known. In 'Baron Solemacher' recessive alleles of an unknown repressor, SEASONAL FLOWERING LOCUS (SFL), are responsible for continuous flowering habit. Although flower induction has a central effect on the cropping potential, the molecular control of flowering in strawberries has not been studied and the genetic flowering pathways are still poorly understood. The comparison of everbearing and short-day genotypes of wild strawberry could facilitate our understanding of fundamental molecular mechanisms regulating perennial growth cycle in plants.     

Signature-Tagged Mutagenesis in a Chicken Infection Model Leads to the Identification of a Novel Avian Pathogenic Escherichia coli Fimbrial Adhesin

The extraintestinal pathogen, avian pathogenic E. coli (APEC), known to cause systemic infections in chickens, is responsible for large economic losses in the poultry industry worldwide. In order to identify genes involved in the early essential stages of pathogenesis, namely adhesion and colonization, Signature-tagged mutagenesis (STM) was applied to a previously established lung colonization model of infection by generating and screening a total of 1,800 mutants of an APEC strain IMT5155 (O2:K1:H5; Sequence type complex 95). The study led to the identification of new genes of interest, including two adhesins, one of which coded for a novel APEC fimbrial adhesin (Yqi) not described for its role in APEC pathogenesis to date. Its gene product has been temporarily designated ExPEC Adhesin I (EA/I) until the adhesin-specific receptor is identified. Deletion of the ExPEC adhesin I gene resulted in reduced colonization ability by APEC strain IMT5155 both in vitro and in vivo. Furthermore, complementation of the adhesin gene restored its ability to colonize epithelial cells in vitro. The ExPEC adhesin I protein was successfully expressed in vitro. Electron microscopy of an afimbriate strain E. coli AAEC189 over-expressed with the putative EA/I gene cluster revealed short fimbrial-like appendages protruding out of the bacterial outer membrane. We observed that this adhesin coding gene yqi is prevalent among extraintestinal pathogenic E. coli (ExPEC) isolates, including APEC (54.4%), uropathogenic E. coli (UPEC) (65.9%) and newborn meningitic E. coli (NMEC) (60.0%), and absent in all of the 153 intestinal pathogenic E. coli strains tested, thereby validating the designation of the adhesin as ExPEC Adhesin I. In addition, prevalence of EA/I was most frequently associated with the B2 group of the EcoR classification and ST95 complex of the multi locus sequence typing (MLST) scheme, with evidence of a positive selection within this highly pathogenic complex. This is the first report of the newly identified and functionally characterized ExPEC adhesin I and its significant role during APEC infection in chickens.     

Contrasting patterns of tree mortality in late‐successional Picea abies stands in two areas in northern Fennoscandia

Question: What are tree mortality rates and how and why do they vary in late‐successional Picea abies‐dominated forests? Do observed tree mortality patterns allow comparative assessment of models of long‐term stand development? Location: Northern boreal Fennoscandia. Methods: We measured stand structure in 10 stands in two different areas. We determined age distributions and constructed a chronology of tree deaths by cross‐dating the years of death of randomly sampled dead trees. Results: The stands in the two areas had contrasting tree age distributions, despite similar live tree structure. In one area, stands were relatively even‐aged and originated following a stand‐replacing fire 317 years earlier. The stands in the second area had an uneven age structure and virtually no signs of past fires, suggesting a very long period since the last major disturbance. The younger stands were characterized by a high mortality rate and inter‐annual variation, which we attributed to senescence of the relatively even‐aged stands approaching the maximum age of P. abies. In contrast, the tree mortality rates in the older stands were low and relatively stable. Conclusions: Patterns of tree mortality were, to a large extent, dependent on the time since the last stand‐replacing disturbance, suggesting that northern boreal P. abies stands eventually reach a shifting mosaic state maintained through small‐scale dynamics, but the time needed to reach this state appears to be lengthy; even 300 years after a forest fire stands showed changes in patterns of tree mortality that were related to the developmental stage of the stands.     

Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues

To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 degrees C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 degrees C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.     

Trypsins and their role in carcinoma growth

There are more than 100 distinct types of cancer, and subtypes can be found within specific organs. Cancer progression is a complex multi-step process. These steps reflect alterations that drive the progressive transformation of normal cells into highly malignant ones. One critical step in tumor growth and invasion is the proteolytic processing of the extracellular matrix environment. The degradation of the extracellular matrix not only enables cell migration, invasion, and metastasis formation, but also affects cell behavior in multiple ways; on one hand by cleaving extracellular matrix bound growth factors and on the other hand by inhibiting angiogenesis into the tumor by liberating cryptic endogenous inhibitors of angiogenesis. Serine proteases and matrix metalloproteases are families of proteolytic enzymes involved in physiological and pathological extracellular matrix and basement membrane processing. In this review, we will focus on the role and activation of trypsinogens, a family of serine proteases, in cancer progression.     

A phage display screen and binding studies with acetylated low density lipoprotein provide evidence for the importance of the scavenger receptor cysteine-rich (SRCR) domain in the ligand-binding function of MARCO

MARCO is a class A scavenger receptor capable of binding both gram-negative and -positive bacteria. Using the surface plasmon resonance technique, we show here that a recombinant, soluble form of MARCO, sMARCO, binds the major gram-negative and -positive bacterial surface components, lipopolysaccharide and lipoteichoic acid. Yet, the interaction of these two polyanions with sMARCO is of much lower affinity than that of polyinosinic acid, a polyanionic inhibitor of bacterial binding to MARCO. To further elucidate the ligand-binding functions of MARCO, we performed a phage display screen with sMARCO. The screening resulted in the enrichment of only a handful of phage clones. Contrary to expectations, no polyanionic peptides, but only those with a predominantly hydrophobic nature, were enriched. One peptide, VRWGSFAAWL, was displayed on two-thirds of the phages recovered after four rounds of screening. The VRWGSFAAWL phage-sMARCO interaction had significantly slower dissociation kinetics than that between sMARCO and lipopolysaccharide or lipoteichoic acid. Further work with this phage, and the second most enriched phage, displaying the peptide RLNWAWWLSY, demonstrated that both peptides bind to the SRCR domain of MARCO, and that they probably bind to the same site. Data base searches suggested that the VRWGSFAAWL peptide represents complement component C4, but we could not convincingly confirm this suggestion. A study with chimeric scavenger receptors indicated that even minor sequence changes in the MARCO scavenger receptor cysteine-rich (SRCR) domain can have profound effects on the binding of the prototypic scavenger receptor ligand, acetylated low density lipoprotein. As shown by differential binding of glutathione S-transferase-VR-WGSFAAWL, these differences were very likely due to conformational changes. These findings led to experiments that demonstrated a crucial role of the SRCR domain for acetylated low density lipoprotein binding in MARCO. Thus, our results strengthen the notion that the SRCR domain is the major ligand-binding domain in MARCO. Furthermore, they suggest that the domain may contain multiple ligand-binding sites.     

Effect of Nutrient Loading on Bacterioplankton Community Composition in Lake Mesocosms

Changes in bacterioplankton community composition were followed in mesocosms set up in the littoral of Lake Vesijärvi, southern Finland, over two summers. Increasing nitrogen and phosphorus concentrations in the mesocosms represented different trophic states, from mesotrophic to hypertrophic. In 1998, the mesocosms were in a turbid state with a high biomass of phytoplankton, whereas in 1999, macrophytes proliferated and a clear-water state prevailed. The bacterial communities in the mesocosms also developed differently, as shown by denaturing gradient gel electrophoresis profiling of partial 16S rRNA gene fragments and by nonmetric multidimensional scaling analysis. In 1998, nutrient treatments affected the diversity and clustering of bacterial communities strongly, but in 1999, the bacterial communities were less diversified and not clearly affected by treatments. Canonical correspondence analysis indicated that bacterioplankton communities in the mesocosms were influenced by environmental physicochemical variables linked to the increasing level of eutrophication. Nitrogen concentration correlated directly with the bacterioplankton composition. In addition, the high nutrient levels had indirect effects through changes in the biomass and composition of phyto- and zooplankton. Sequencing analysis showed that the dominant bacterial divisions remained the same, but the dominant phylotypes changed during the 2-year period. The occurrence of Verrucomicrobia correlated with more eutrophic conditions, whereas the occurrence of Actinobacteria correlated with less eutrophic conditions.