Bioinformatics tools and database resources for systems genetics analysis in mice--a short review and an evaluation of future needs

During a meeting of the SYSGENET working group 'Bioinformatics', currently available software tools and databases for systems genetics in mice were reviewed and the needs for future developments discussed. The group evaluated interoperability and performed initial feasibility studies. To aid future compatibility of software and exchange of already developed software modules, a strong recommendation was made by the group to integrate HAPPY and R/qtl analysis toolboxes, GeneNetwork and XGAP database platforms, and TIQS and xQTL processing platforms. R should be used as the principal computer language for QTL data analysis in all platforms and a 'cloud' should be used for software dissemination to the community. Furthermore, the working group recommended that all data models and software source code should be made visible in public repositories to allow a coordinated effort on the use of common data structures and file formats.     

The effect of three years of TNF alpha blocking therapy on markers of bone turnover and their predictive value for treatment discontinuation in patients with ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction

Statins (hydroxymethylglutaryl coenzyme A reductase inhibitors) are effective in reducing the risk of cardiovascular morbidity and mortality in patients with hyperlipidemia, hypertension, or type II diabetes. Next to their cholesterol-lowering activity, statins have immunomodulatory properties. Based on these properties, we hypothesized that statin use may eventually lead to dysregulation of immune responses, possibly resulting in autoimmunity. We have recently shown in an observational study that statin use was associated with an increased risk of developing rheumatoid arthritis. Our objective was to investigate whether a causal relationship could be established for this finding.

Methods

The mouse collagen type II (CII)-induced arthritis (CIA) model was used, with immunization, challenge, and euthanasia at days 0, 21, and 42, respectively. Statins were given orally before (day -28 until day 21) or after (day 21 until day 42) CIA induction. Atorvastatin (0.2 mg/day) or pravastatin (0.8 mg/day) was administered. Arthritis was recorded three times a week. Serum anti-CII autoantibodies and cytokines in supernatants from Concanavalin-A-stimulated lymph node cells and CII-stimulated spleen cells were measured.

Results

Statin administration accelerated arthritis onset and resulted in 100% arthritic animals, whereas only seven out of 12 nonstatin control animals developed arthritis. Atorvastatin administration after CIA induction resulted in earlier onset than atorvastatin administration before induction, or than pravastatin administration before or after induction. The arthritic score of animals given pravastatin before CIA induction was similar to that of the nonstatin controls, whereas the other groups that received statins showed higher arthritic scores. Atorvastatin administration, especially before CIA induction, increased anti-CII autoantibody production. IL-2 and IL-17 production by lymph node and spleen cells was higher in CIA animals than in PBS controls, but was not affected by statin administration. While IFN?? production was not affected by CIA induction, atorvastatin administration before CIA induction increased the production of this cytokine.

Conclusion

These data support previous results from our observational studies, indicating a role for statins in the induction of autoimmunity.     

Pre-steady state kinetic studies on the microsecond time scale of the laccase from Trametes versicolor

Laccase from Trametes versicolor reduces dioxygen to water. The enzyme is used in green chemistry applications such as the selective oxidation of alcohols in the presence of a suitable mediator (TEMPO) or in biofuel cells. We studied the catalytic mechanism of the enzyme by the stopped-flow and our newly developed rapid-mixing rapid sampling method, which has an experimental dead time of 75 ± 15 μs. Equilibrium and kinetic analyses yielded a reduction potential of 717 ± 5 mV for Type 1 copper center. EPR and low-temperature UV-Vis spectroscopy indicate that oxidation of the blue copper center and OO bond splitting occur within 100 μs, without detectable formation of a peroxide intermediate. These results indicate a rapid internal electron transfer between the various copper centers (>25.000/s) and rapid binding of O₂ (k_on > 5 × 10⁷ M⁻¹ s⁻¹). Mechanistic aspects of the catalytic cycle are shortly discussed.     

Pyruvate relieves the necessity of high induction levels of catalase and enables Campylobacter jejuni to grow under fully aerobic conditions

Aims:  Several cases of campylobacteriosis reported worldwide seemingly conflict with the strict growth requirements and sensitivity to environmental stress of Campylobacter jejuni. In this study, the need for a micro‐aerobic environment [dissolved oxygen tension (DOT): 0·1–90%; 100% air saturation)] and the adaptive responses to oxygen stress were studied. Methods and Results:  The growth of C. jejuni in continuous culture was assessed under different DOT in the presence or absence of pyruvate. In a medium without pyruvate, continuous cultures of C. jejuni showed typically micro‐aerobic behaviour and cells were unable to grow under fully aerobic conditions. However in the presence of pyruvate (25 mmol l^?1), continuous cultures of C. jejuni were able to grow in a broad DOT range, varying from 0·1% to at least 90%, and the catalase activity was decreased. Conclusions:  Addition of pyruvate results in the decrease in the concentration of hydrogen peroxide, which enables C. jejuni to grow aerobically. Significance and Impact of the Study:  New information on the oxidative physiology of C. jejuni and its ability to grow aerobically in media supplemented with pyruvate is presented.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

Enzyme-catalysed deprotection of N-acetyl and N-formyl amino acids

To investigate the full potential of hydrolases for the removal of two amine-protecting groups, 15 different, commercially available lipases, acylases, proteases and esterases were studied for the hydrolyses of N-acetyl and N-formyl protecting groups. In addition to the well-known acylases from porcine kidney and Aspergillus melleus, this screening revealed that porcine liver esterase and the lipases from Rhizomucor miehei and Pseudomonas stutzeri are also catalysts for the hydrolysis of N-acetylalanine. The activity of lipases in this reaction was unexpected, since lipases are commonly believed not to hydrolyse amides. In addition, from these 15 enzymes, three were found to be active in the hydrolysis of N-formylalanine, i.e. porcine liver esterase and the two acylases. This is the first example where esterase is employed to deprotect N-formyl amides.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.