Whole-body cryotherapy for the treatment of rheumatoid arthritis: a monocentric, single-blinded, randomised controlled trial.

OBJECTIVES: To evaluate effects of whole-body cryotherapy (WBC) in rheumatoid arthritis (RA). METHODS: Patients with active RA undergoing a 16-day multimodal rheumatologic complex treatment were randomly assigned to either WBC (6 applications in 14 days at -130°C for 3 min) or no treatment. The primary outcome was the difference between groups in pain on a numerical rating scale after intervention. Secondary outcomes assessed effects on i) disease activity, ii) functional capacity, iii) cytokine levels, and iv) use of analgesics. RESULTS: A total of 56 RA patients completed the trial (intervention group [IG]: 31 patients, control group [CG]: 25 patients). The mean change (± standard error) in pain after intervention was -2 in the IG (95% confidence interval [CI] -2.75 to -1.31, p<0.001) and -0.88 (95% CI -1.43 to -0.33, p=0.003) in the CG, with a baseline-adjusted between-group difference of -1.31 ± 0.4 (95% CI -2.1 to -0.53; p=0.002). Pain at the 12-week follow-up visit remained significantly below baseline values in the IG. Disease activity and functional capacity showed statistically and clinically meaningful improvement after intervention but were not significant at the 12-week follow up. TNF and IL-6 levels changed significantly in the IG. Eighteen of 31 (58%) patients of the IG reduced or discontinued analgesics at the 12-week follow-up. No WBC-related side effects were reported. CONCLUSIONS: WBC in RA reduces pain and disease activity significantly and in a clinically meaningful manner, resulting in a reduction of analgesics. These effects are potentially based on a change in cytokine levels.

Protein resonance assignment by BSH-CP-based 3D solid-state NMR experiments: A practical guide.

Solid-state NMR (ssNMR) spectroscopy has evolved into a powerful method to obtain structural information and to study the dynamics of proteins at atomic resolution and under physiological conditions. The method is especially well suited to investigate insoluble and noncrystalline proteins that cannot be investigated easily by X-ray crystallography or solution NMR. To allow for detailed analysis of ssNMR data, the assignment of resonances to the protein atoms is essential. For this purpose, a set of three-dimensional (3D) spectra needs to be acquired. Band-selective homo-nuclear cross-polarization (BSH-CP) is an effective method for magnetization transfer between carbonyl carbon (CO) and alpha carbon (CA) atoms, which is an important transfer step in multidimensional ssNMR experiments. This tutorial describes the detailed procedure for the chemical shift assignment of the backbone atoms of 13 C-15 N-labeled proteins by BSH-CP-based 13 C-detected ssNMR experiments. A set of six 3D experiments is used for unambiguous assignment of the protein backbone as well as certain side-chain resonances. The tutorial especially addresses scientists with little experience in the field of ssNMR and provides all the necessary information for protein assignment in an efficient, time-saving approach.

Dynamic carbon flux network of a diverse marine microbial community.

The functioning of microbial ecosystems has important consequences from global climate to human health, but quantitative mechanistic understanding remains elusive. The components of microbial ecosystems can now be observed at high resolution, but interactions still have to be inferred e.g., a time-series may show a bloom of bacteria X followed by virus Y suggesting they interact. Existing inference approaches are mostly empirical, like correlation networks, which are not mechanistically constrained and do not provide quantitative mass fluxes, and thus have limited utility. We developed an inference method, where a mechanistic model with hundreds of species and thousands of parameters is calibrated to time series data. The large scale, nonlinearity and feedbacks pose a challenging optimization problem, which is overcome using a novel procedure that mimics natural speciation or diversification e.g., stepwise increase of bacteria species. The method allows for curation using species-level information from e.g., physiological experiments or genome sequences. The product is a mass-balancing, mechanistically-constrained, quantitative representation of the ecosystem. We apply the method to characterize phytoplankton-heterotrophic bacteria interactions via dissolved organic matter in a marine system. The resulting model predicts quantitative fluxes for each interaction and time point (e.g., 0.16 µmolC/L/d of chrysolaminarin to Polaribacter on April 16, 2009). At the system level, the flux network shows a strong correlation between the abundance of bacteria species and their carbon flux during blooms, with copiotrophs being relatively more important than oligotrophs. However, oligotrophs, like SAR11, are unexpectedly high carbon processors for weeks into blooms, due to their higher biomass. The fraction of exudates (vs. grazing/death products) in the DOM pool decreases during blooms, and they are preferentially consumed by oligotrophs. In addition, functional similarity of phytoplankton i.e., what they produce, decouples their association with heterotrophs. The methodology is applicable to other microbial ecosystems, like human microbiome or wastewater treatment plants.

Crystal structures of the selenoprotein glutathione peroxidase 4 in its apo form and in complex with the covalently bound inhibitor ML162.

Wild-type human glutathione peroxidase 4 (GPX4) was co-expressed with SBP2 (selenocysteine insertion sequence-binding protein 2) in human HEK cells to achieve efficient production of this selenocysteine-containing enzyme on a preparative scale for structural biology. The protein was purified and crystallized, and the crystal structure of the wild-type form of GPX4 was determined at 1.0 Šresolution. The overall fold and the active site are conserved compared with previously determined crystal structures of mutated forms of GPX4. A mass-spectrometry-based approach was developed to monitor the reaction of the active-site selenocysteine Sec46 with covalent inhibitors. This, together with the introduction of a surface mutant (Cys66Ser), enabled the crystal structure determination of GPX4 in complex with the covalent inhibitor ML162 [(S)-enantiomer]. The mass-spectrometry-based approach described here opens the path to further co-complex crystal structures of this potential cancer drug target in complex with covalent inhibitors.

Light-driven formation of manganese oxide by today's photosystem II supports evolutionarily ancient manganese-oxidizing photosynthesis.

Water oxidation and concomitant dioxygen formation by the manganese-calcium cluster of oxygenic photosynthesis has shaped the biosphere, atmosphere, and geosphere. It has been hypothesized that at an early stage of evolution, before photosynthetic water oxidation became prominent, light-driven formation of manganese oxides from dissolved Mn(2+) ions may have played a key role in bioenergetics and possibly facilitated early geological manganese deposits. Here we report the biochemical evidence for the ability of photosystems to form extended manganese oxide particles. The photochemical redox processes in spinach photosystem-II particles devoid of the manganese-calcium cluster are tracked by visible-light and X-ray spectroscopy. Oxidation of dissolved manganese ions results in high-valent Mn(III,IV)-oxide nanoparticles of the birnessite type bound to photosystem II, with 50-100 manganese ions per photosystem. Having shown that even today's photosystem II can form birnessite-type oxide particles efficiently, we propose an evolutionary scenario, which involves manganese-oxide production by ancestral photosystems, later followed by down-sizing of protein-bound manganese-oxide nanoparticles to finally yield today's catalyst of photosynthetic water oxidation.

Different structural requirements for functional ion pore transplantation suggest different gating mechanisms of NMDA and kainate receptors.

Although considerable progress has been made in characterizing the physiological function of the high-affinity kainate (KA) receptor subunits KA1 and KA2, no homomeric ion channel function has been shown. An ion channel transplantation approach was employed in this study to directly test if homomerically expressed KA1 and KA2 pore domains are capable of conducting currents. Transplantation of the ion pore of KA1 or KA2 into GluR6 generated perfectly functional ion channels that allowed characterization of those electrophysiological and pharmacological properties that are determined exclusively by the ion pore of KA1 or KA2. This demonstrates for the first time that KA1 and KA2 ion pore domains are intrinsically capable of conducting ions even in homomeric pore assemblies. NMDA receptors, similar to KA1- or KA2-containing receptors, function only as heteromeric complexes. They are composed of NR1 and NR2 subunits, which both are non-functional when expressed homomerically. In contrast to NR1, the homomeric NR2B ion pore failed to translate ligand binding into pore opening when transplanted into GluR6. Similarly, heteromeric coexpression of the ion channel domains of both NR1 and NR2 inserted into GluR6 failed to produce functional channels. Therefore, we conclude that the mechanism underlying the ion channel opening in the obligatorily heterotetrameric NMDA receptors differs significantly from that in the facultatively heterotetrameric alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and KA receptors.

Stargazin interaction with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is critically dependent on the amino acid at the narrow constriction of the ion channel.

The subunit GluR2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subfamily of ionotropic glutamate receptors (GluRs) features a single amino acid at the narrow constriction of the pore loop that is altered from glutamine to arginine by RNA editing. This so-called Q/R site has been shown to play an important role in the determination of the electrophysiological properties of AMPA receptor complexes as well as of trafficking to the plasma membrane. The protein stargazin has also been shown to modulate electrophysiological properties and trafficking to the plasma membrane of AMPA receptors. In this study we examined via a series of mutants of the Q/R site of the AMPA receptor GluR1 whether the amino acid at this position has any influence on the modulatory effects mediated by stargazin. To this end, we analyzed current responses of Q/R site mutants upon application of glutamate and kainate and determined the amount of mutant receptor protein in the plasma membrane in Xenopus oocytes. Desensitization kinetics of several mutants were analyzed in HEK293 cells. We found that the stargazin-mediated decrease in receptor desensitization, the slowing of desensitization kinetics, and the kainate efficacy were all dependent on the amino acid at the Q/R site, whereas the stargazin-mediated increase in trafficking toward the plasma membrane remained independent of this amino acid. We propose that the Q/R site modulates the interaction of stargazin with the transmembrane domains of AMPA receptors via an allosteric mechanism and that this modulation leads to the observed differences in the electrophysiological properties of the receptor.

Ion pore properties of ionotropic glutamate receptors are modulated by a transplanted potassium channel selectivity filter.

The canonical potassium channel selectivity filter motif TVGYG was transplanted into ionotropic glutamate receptors (iGluRs) of the AMPA and NMDA subtype to test whether it renders the iGluRs K(+) selective. The TVGYG motif modulated several ion pore properties of AMPA receptor as well as NMDA receptor mutants, e.g., the intra- and extracellular polyamine block, current/voltage relationships, open channel block by MK801 and Mg(2+), and permeability for divalent cations. However, introduction of the selectivity filter failed to increase the K(+) selectivity of homomeric AMPA and heteromeric NMDA receptor complexes, which may be due to absence of selectivity filter-stabilizing interaction sites in the iGluR pore domain. Our findings indicate that even if glutamate receptors appear to have the intrinsic capacity for K(+) permeability, as is demonstrated by the prokaryotic, glutamate-gated, K(+) selective GluR0, the isolated selectivity filter is not able to confer K(+) permeability to the relatively unselective iGluR cation pore.

Investigation via ion pore transplantation of the putative relationship between glutamate receptors and K+ channels.

The pore domains of ionotropic glutamate receptors (iGluRs) and potassium channels (K(+) channels) show several structural similarities. To test for functional compatibility, we transferred pore regions from prokaryotic, invertebrate, and vertebrate K(+) channels into pharmacologically representative iGluRs and vice versa. Although the chimeric proteins were expressed on the cell surface, only one of 45 pore chimeras showed ion channel function: The kainate receptor subunit GluR6, carrying the pore loop plus adjacent transmembrane domains of the prokaryotic, glutamate-gated, K(+)-selective GluR0, adopted several electrophysiological properties of the donor pore upon pore transplantation. This suggests that, despite structural similarities between iGluR and K(+) channel pores, there is a lack of functional compatibility so that K(+) channel pores cannot be gated by the iGluR gating machinery, and vice versa. However, K(+)-selective pores can be gated in an iGluR sequence environment, given a similar signal transduction mechanism as appears to be present in GluR0.