In vivo biotinylated calpastatin improves the affinity purification of human m-calpain

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies. © 2018 The Author

An ultraviolet B condition that affects growth and defense in Arabidopsis

Ultraviolet B light (UV-B, 280-315 nm) is the shortest wavelength of the solar spectrum reaching the surface of the Earth. It has profound effects on plants, ranging from growth regulation to severe metabolic changes. Low level UV-B mainly causes photomorphogenic effects while higher levels can induce stress, yet these effects tend to overlap. Here we identified a condition that allows growth reduction without obvious detrimental stress in wild type Arabidopsis rosette plants. This condition was used to study the effects of a daily UV-B dose on plant characteristics of UV-B adapted plants in detail. Exploration of the transcriptome of developing leaves indicated downregulation of genes involved in stomata formation by UV-B, while at the same time genes involved in photoprotective pigment biosynthesis were upregulated. These findings correspond with a decreased stomatal density and increased UV-B absorbing pigments. Gene ontology analysis revealed upregulation of defense related genes and meta-analysis showed substantial overlap of the UV-B regulated transcriptome with transcriptomes of salicylate and jasmonate treated as well as herbivore exposed plants. Feeding experiments showed that caterpillars of Spodoptera littoralis are directly affected by UV-B, while performance of the aphid Myzus persicae is diminished by a plant mediated process

The most likely time and place of introduction of BTV8 into belgian ruminants

Peer reviewe

Bluetongue in Belgium, 2006

Bluetongue has emerged recently in Belgium. A bluetongue virus strain was isolated and characterized as serotype 8. Two new real-time reverse transcription–quantitative PCRs (RT-qPCRs) that amplified 2 different segments of bluetongue virus detected this exotic strain. These 2 RT-qPCRs detected infection earlier than a competitive ELISA for antibody detection

Response of Cupriavidus metallidurans CH34 to metals

Cupriavidus metallidurans CH34 displays resistance to a plethora of metals. Its response and underlying genetic determinants are dissected and detailed metal by metal (from arsenic to zinc). An important role for its megaplasmids pMOL28 and pMOL30 is shown, with high level resistance to cadmium, chromate, cobalt, copper, mercury, nickel, lead and zinc mediated by well-known genes for detoxification often accompanied by other functions linked to acute or chronic stress. Nevertheless, metal resistance determinants are also found on the chromid (e.g. to chromate, copper and zinc) as well as on a large genomic island integrated in the chromosome (e.g. to cadmium, lead and mercury), even the core genome participates in certain responses such as to gold or selenium. Next, we summarized the environmental applications, which were developed based on the knowledge gained by studying these different determinants, and in particular biosensors and soil and water bioremediation. Finally, the general transcriptional response of C. metallidurans to sixteen different metals supplied at different concentrations (including acute stress) is discussed within the framework of its intricate regulatory network

Interplays between copper and Mycobacterium tuberculosis GroEL1

The recalcitrance of pathogenic Mycobacterium tuberculosis, the agent of tuberculosis, to eradication is due to various factors allowing bacteria to escape from stress situations. The mycobacterial chaperone GroEL1, overproduced after macrophage entry and under oxidative stress, could be one of these key players. We previously reported that GroEL1 is necessary for the biosynthesis of phthiocerol dimycocerosate, a virulence-associated lipid and for reducing antibiotic susceptibility. In the present study, we showed that GroEL1, bearing a unique C-terminal histidine-rich region, is required for copper tolerance during Mycobacterium bovis BCG biofilm growth. Mass spectrometry analysis demonstrated that GroEL1 displays high affinity for copper ions, especially at its C-terminal histidine-rich region. Furthermore, the binding of copper protects GroEL1 from destabilization and increases GroEL1 ATPase activity. Altogether, these findings suggest that GroEL1 could counteract copper toxicity, notably in the macrophage phagosome, and further emphasizes that M. tuberculosis GroEL1 could be an interesting antitubercular target

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure