Variability in synovial inflammation in rheumatoid arthritis investigated by microarray technology

In recent years microarray technology has been used increasingly to acquire knowledge about the pathogenic processes involved in rheumatoid arthritis. The present study investigated variations in gene expression in synovial tissues within and between patients with rheumatoid arthritis. This was done by applying microarray technology on multiple synovial biopsies obtained from the same knee joints. In this way the relative levels of intra-patient and inter-patient variation could be assessed. The biopsies were obtained from 13 different patients: 7 by orthopedic surgery and 6 by rheumatic arthroscopy. The data show that levels of heterogeneity varied substantially between the biopsies, because the number of genes found to be differentially expressed between pairs of biopsies from the same knee ranged from 6 to 2,133. Both arthroscopic and orthopedic biopsies were examined, allowing us to compare the two sampling methods. We found that the average number of differentially expressed genes between biopsies from the same patient was about three times larger in orthopedic than in arthroscopic biopsies. Using a parallel analysis of the tissues by immunohistochemistry, we also identified orthopedic biopsies that were unsuitable for gene expression analysis of synovial inflammation due to sampling of non-inflamed parts of the tissue. Removing these biopsies reduced the average number of differentially expressed genes between the orthopedic biopsies from 455 to 171, in comparison with 143 for the arthroscopic biopsies. Hierarchical clustering analysis showed that the remaining orthopedic and arthroscopic biopsies had gene expression signatures that were unique for each patient, apparently reflecting patient variation rather than tissue heterogeneity. Subsets of genes found to vary between biopsies were investigated for overrepresentation of biological processes by using gene ontology. This revealed representative 'themes' likely to vary between synovial biopsies affected by inflammatory disease.     

Effects of landscape composition and substrate availability on saproxylic beetles in boreal forests: a study using experimental logs for monitoring assemblages

Intensive forestry practises in the Swedish landscape have led to the loss and fragmentation of stable old‐growth habitats. We investigated relationships between landscape composition at multiple scales and the composition of saproxylic beetle assemblages in nine clear‐cut, mature managed and old‐growth spruce‐dominated forest stands in the central boreal zone of Sweden. We set out fresh spruce and birch logs and created spruce snags in 2001–2002 to experimentally test the effects of coarse woody debris (CWD) type and forest management on the composition of early and late successional, and red‐listed saproxylic beetle assemblages. We examined effects of CWD availability at 100 m, and landscape composition at 1 and 10 km on saproxylic beetle abundances. Additionally, we tested whether assemblage similarity decreased with increasing distance between sites. We collected beetles from the experimental logs using eclector and window traps in four periods during 2003. CWD was measured and landscape composition data was obtained from maps of remotely sensed data. The composition of saproxylic beetles differed among different CWD substrates and between clear‐cuts and the older stand types, however differences between mature managed and old‐growth forests were significant only for red‐listed species. Assemblage similarities for red‐listed species on clear‐cuts were more different at greater distances apart, indicating that they have more localised distributions. CWD availability within 100 m of the study sites was rarely important in determining the abundance of species, suggesting that early successional saproxylic beetles can disperse further than this distance. At a larger scale, a large area of suitable stand types within both 1 and 10 km resulted in greater abundances in the study sites for several common and habitat‐specific species. The availability of suitable habitat at scales of 1–10 km is thus likely to be important in the survival of many saproxylic species in forestry‐fragmented areas.     

Evolution of immunity and host chromosome integration site of P2-like coliphages

The amount and distribution of variation in the genomic region containing the genes in the lytic-lysogenic genetic switch and the sequence that determines the integration site into the host chromosome were analyzed for 38 P2-like phages from Escherichia coli. The genetic switch consists of two convergent mutually exclusive promoters, Pe and Pc, and two repressors, C and Cox. The immunity repressor C blocks the early Pe promoter, leading to the establishment of lysogeny. The Cox repressor blocks expression of Pc, allowing lytic growth. Phylogenetic analyses showed that the C and Cox proteins were distributed into seven distinct classes. The phylogenetic relationship differed between the two proteins, and we showed that homologous recombination plays a major role in creating alterations in the genetic switch, leading to new immunity classes. Analyses of the host integration site for these phages resulted in the discovery of a previously unknown site, and there were at least four regular integration sites. Interestingly, we found no case where phages of the same immunity class had different host attachment sites. The evolution of immunity and integration sites is complex, since it involves interactions both between the phages themselves and between phages and hosts, and often, both regulatory proteins and target DNA must change.     

Unravelling the evolution of neural stem cells in arthropods: Notch signalling in neural stem cell development in the crustacean Daphnia magna

The genetic regulatory networks controlling major developmental processes seem to be conserved in bilaterians regardless of an independent or a common origin of the structures. This has been explained by the employment of a genetic toolkit that was repeatedly used during bilaterian evolution to build the various forms and body plans. However, it is not clear how genetic networks were incorporated into the formation of novel structures and how homologous genes can regulate the disparate morphological processes. Here we address this question by analysing the role of Notch signalling, which is part of the bilaterian toolkit, in neural stem cell evolution in arthropods. Within arthropods neural stem cells have evolved in the last common ancestor of insects and crustaceans (Tetraconata). We analyse here for the first time the role of Notch signalling in a crustacean, the branchiopod Daphnia magna, and show that it is required in neural stem cells for regulating the time of neural precursor production and for binary cell fate decisions in the ventral neuroectoderm. The function of Notch signalling has diverged in the ventral neuroectoderm of insects and crustaceans accompanied by changes in the morphogenetic processes. In the crustacean, Notch controlled mechanisms of neuroblast regulation have evolved that are surprisingly similar to vertebrates and thus present a remarkable case of parallel evolution. These new data on a representative of crustaceans complete the arthropod data set on Notch signalling in the nervous system and allow for reconstructing how the Notch signalling pathway has been co-opted from pre-existing structures to the development of the evolving neural stem cells in the Tetraconata ancestor.     

Single-minded and the evolution of the ventral midline in arthropods

Glypicans are multifunctional proteoglycans with regulatory roles in several intercellular signaling pathways. Here, we examine the functional requirements for glypican regulation of bone morphogenetic protein (BMP)-mediated body length in C. elegans. We provide evidence that two parts of C. elegans glypican LON-2 can independently inhibit BMP signaling in vivo: the N-terminal furin protease product and the C-terminal region containing heparan sulfate attachment sequences. While the C-terminal protease product is dispensable for LON-2 minimal core protein activity, it does affect the localization of LON-2. Cleavage of LON-2 into two parts at the conserved furin protease site is not required for LON-2 to inhibit BMP-like signaling. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not absolutely required for LON-2 activity. Finally, we show that an RGD protein-protein interaction motif in the LON-2 N-terminal domain is necessary for LON-2 core protein activity, suggesting that LON-2 inhibits BMP signaling by acting as a scaffold for BMP and an RGD-binding protein.     

Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background

Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.

Methodology/Principal Findings

In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.

Conclusions/Significance

We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers.     

Novel thioamide derivatives as neutral CB1 receptor antagonists

A novel class of cannabinoid-1 (CB1) receptor antagonists for the treatment of obesity is presented. The carboxamide linker in a set of 5,6-diaryl-pyrazine-2-amide derivatives was transformed into the corresponding thioamide, by using a one-pot synthesis. The structural series of thioamides not only showed retained CB1 potency (below 10 nM), but also showed improved solubility. In addition, the neutral antagonist 2c significantly reduced body weight in cafeteria diet obese mice.     

TagGD: Fast and Accurate Software for DNA Tag Generation and Demultiplexing

Multiplexing is of vital importance for utilizing the full potential of next generation sequencing technologies. We here report TagGD (DNA-based Tag Generator and Demultiplexor), a fully-customisable, fast and accurate software package that can generate thousands of barcodes satisfying user-defined constraints and can guarantee full demultiplexing accuracy. The barcodes are designed to minimise their interference with the experiment. Insertion, deletion and substitution events are considered when designing and demultiplexing barcodes. 20,000 barcodes of length 18 were designed in 5 minutes and 2 million barcoded Illumina HiSeq-like reads generated with an error rate of 2% were demultiplexed with full accuracy in 5 minutes. We believe that our software meets a central demand in the current high-throughput biology and can be utilised in any field with ample sample abundance. The software is available on GitHub (https://github.com/pelinakan/UBD.git).     

Expressed Prostate Secretions in the Study of Human Papillomavirus Epidemiology in the Male

Introduction

Exploring different sampling sites and methods is of interest for studies of the epidemiology of HPV infections in the male. Expressed prostate secretions (EPS) are obtained during digital rectal examination (DRE), a daily routine urological diagnostic procedure, following massage of the prostate.

Materials and Methods

Urethral swabs and EPS samples were obtained from a consecutive sample of 752 men (mean age 32.4 years; median life-time sex partners 34) visiting urology outpatient clinics in St. Petersburg, Russia and tested for HPV DNA by general primer PCR, followed by genotyping using Luminex.

Results

Overall, 47.9% (360/752) of men were HPV-positive, with 42.0% (316/752) being positive for high-risk (HR-) HPV and 12.6% (95/752) for multiple HPV types. HPV-positivity in the EPS samples was 32.6% (27.7% HR-HPV) and in the urethral samples 25.9% (24.5% HR-HPV). 10.6% were HPV positive in both EPS and urethral samples. 6.4% had the same HPV-type in both EPS and urethral samples. 10.6% were HPV positive in both EPS and urethral samples. 6.4% had the same HPV-type in both EPS and urethral samples. The concordance between the urethral samples and EPS was 62.5% (470/752), with 80 cases double positive and 390 cases double negative in both sites. The sensitivity of urethral samples for overall HPV detection was 54.2% (195/360). Compared to analysis of urethral samples only, the analysis of EPS increased the HPV prevalence in this population with 26.2%.

Conclusion

EPS represent informative sampling material for the study of HPV epidemiology in the male.