Detecting peroxiredoxin hyperoxidation by one-dimensional isoelectric focusing

The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation

Lack of an efficient endoplasmic reticulum-localized recycling system protects peroxiredoxin IV from hyperoxidation

Typical 2-cys peroxiredoxins are required to remove hydrogen peroxide from several different cellular compartments. Their activity can be regulated by hyperoxidation and consequent inactivation of the active site peroxidatic cysteine. Here we have developed a simple assay to quantify the hyperoxidation of peroxiredoxins. Hyperoxidation of peroxiredoxins can only occur efficiently in the presence of a recycling system usually based on thioredoxin and thioredoxin reductase. We demonstrate that there is a marked difference in the sensitivity of the endoplasmic reticulum-localized peroxiredoxin to hyperoxidation compared to either the cytosolic or mitochondrial enzymes. Each enzyme is equally sensitive to hyperoxidation in the presence of a robust recycling system. Our results demonstrate that the peroxiredoxin IV recycling in the ER is much less efficient than in the cytosol or mitochondria leading to the protection of peroxiredoxin IV from hyperoxidation

Structure-function studies of PRX III, a mitochondrial typical 2-CYS peroxiredoxin

The peroxiredoxuis (Prxs) are a ubiquitous family of antioxidant enzymes that regulate intracellular levels of H2O2 where they are implicated in both tissue protection against oxidative stress and H2O2-niediated signalling pathways (Wood et al., 2003). This thesis describe our results on the structure-function studies of PrxIII, a mitochondrial typical 2-Cys peroxiredoxin. Bovine PrxIII was cloned previously in our laboratory; however PrxIII requires its cognate partners, mammalian mitochondrial thioredoxin (Trx2) and mammalian mitochondrial thioredoxin reductase (TRR2), to reconstitute the complete antioxidant defence system. To establish a direct in vitro assay for PrxIII, Trx2 and TRR2 were cloned, overexpressed and purified in this study. As TRR2 is a selenocysteine (SeCys) protein, a suitable selenocysteine insert sequence (SECIS) for the translation of its penultimate SeCys codon was introduced by incorporating it into the reverse primer for PCR. A combination of different approaches was used for the successful overexpression of active TRR2. Overexpression in modified rich LB media at 15°C in the presence of low IPTG concentrations gave good overexpression of soluble enzyme. Moreover, the addition of the SECIS at the C-terminal of the insert, in the presence of 1 nM Na2SeO3 and co-expression of the SelABC plasmid ensured an optimal supply of the relevant tRNA, tRNA synthase and elongation factor for translation of the UGA SeCys codon. Assays showed that NADPH-linked oxidation needed the presence of all three enzymes to reduce H2O2. PrxIII was also shown to reduce other organic peroxides, although with lower activity. Cys47 and Cysl68, but not Cys66, proved to be crucial for peroxiredoxin activity. Interestingly, at high H2O2 concentrations in the non- physiological range, TRR2 also had the capacity to reduce H2O2 directly in an NADPH-dependent manner. PrxIII is also shown to be susceptible to overoxidation and loses peroxidase activity at increased H2O2 levels in the range 50μM to 1mM. This was monitored by SDS-PAGE analysis of partially or fully overoxidised forms of H2O2-mediated PrxIII and PrxIII pathway assays. Gel filtration chromatography was used to determine under which conditions the PrxIII dodecamer would dissociate into dimers. The results show that redox state, protein concentration and the N-terminal His-tag all affect the oligomerization of PrxIII. The crystal structure of the PrxIII C168S mutant from bovine mitochondria has been determined at a resolution of 3.3 angstroms. The structure reveals that the toroid is composed of 12 (not 10) monomers with a 6(2,2) symmetry. Each ring has an external diameter of 150 angstroms and encompasses a central cavity 70 angstroms in width. Surprisingly, two PrxIII rings are mechanically interlocked in the crystal to form a protein catenane. Interestingly, the catenated form represents only a small proportion (3-5%) of the total PrxIII population, as observed by electronic microscopy studies at dilute concentration (10-50μg/ml). Preliminary analytical ultracentrifugation data suggest that 2-ring catenane formation is concentration dependent. A general model illustrating catenane formation arising from polar contacts between two basic dimeric units is described. It is not clear whether the catenated form of PrxIII has any physiological function. However, the observation that Prxs can protect cells from heat shock in a peroxidase-independent process might provide new insights into possible novel functions

THE EFFECT OF 12 WEEKS CIRCUIT-TRAINING ON HEEL CONTACT VELOCITY AND REACTION TIME IN ELDERLY WOMEN

The purpose of this study was to determine the effects of circuit-training on heel contact velocity (HCV) during walking and reaction time (RT) in community-dwelling elderly women. Subjects were 20 healthy independent elderly women who participated in circuittraining which consisted of posture control, strength training and walking training for 12 weeks. Study outcomes included gait test, reaction time test, and 30-s chair stand test. RT and HCV were decreased significantly. The times of the 30-s chair stand test was increased significantly after training. These findings suggest that 12 weeks of circuittraining may attenuate the risks of slips and slip-initiated falls during walking in community-dwelling elderly women

Structure and electron-transfer pathway of the human methionine sulfoxide reductase MsrB3

Introduction: The post-translational oxidation of methionine to methionine sulfoxide is a reversible process, enabling repair of oxidative damage to proteins and the use of sulfoxidation as a regulatory switch. Methionine sulfoxide reductases catalyze the stereospecific reduction of methionine sulfoxide. One of the mammalian methionine sulfoxide reductases, MsrB3, has a signal sequence for entry into the endoplasmic reticulum (ER). In the ER, MsrB3 is expected to encounter a distinct redox environment compared to its paralogs in the cytosol, nucleus, and mitochondria. Aims: We sought to determine the location and arrangement of MsrB3 redox-active cysteines, which may couple MsrB3 activity to other redox events in the ER. Results: We determined the human MsrB3 structure using X-ray crystallography. The structure revealed that a disulfide bond near the protein amino terminus is distant in space from the active site. Nevertheless, biochemical assays showed that these amino-terminal cysteines are oxidized by the MsrB3 active site after its reaction with methionine sulfoxide. Innovation: This study reveals a mechanism to shuttle oxidizing equivalents from the primary MsrB3 active site toward the enzyme surface, where they would be available for further dithiol-disulfide exchange reactions. Conclusion: Conformational changes must occur during the MsrB3 catalytic cycle to transfer oxidizing equivalents from the active site to the amino-terminal redox-active disulfide. The accessibility of this exposed disulfide may help couple MsrB3 activity to other dithiol/disulfide redox events in the secretory pathway

A Solution to Co-occurrence Bias: Attributes Disentanglement via Mutual Information Minimization for Pedestrian Attribute Recognition

Recent studies on pedestrian attribute recognition progress with either explicit or implicit modeling of the co-occurrence among attributes. Considering that this known a prior is highly variable and unforeseeable regarding the specific scenarios, we show that current methods can actually suffer in generalizing such fitted attributes interdependencies onto scenes or identities off the dataset distribution, resulting in the underlined bias of attributes co-occurrence. To render models robust in realistic scenes, we propose the attributes-disentangled feature learning to ensure the recognition of an attribute not inferring on the existence of others, and which is sequentially formulated as a problem of mutual information minimization. Rooting from it, practical strategies are devised to efficiently decouple attributes, which substantially improve the baseline and establish state-of-the-art performance on realistic datasets like PETAzs and RAPzs. Code is released on https://github.com/SDret/A-Solution-to-Co-occurence-Bias-in-Pedestrian-Attribute-Recognition.Comment: Accepted in IJCAI2

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so‐called non‐native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non‐native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non‐native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non‐native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway

PO-256 Influences of Exercise on Circulating Irisin in Overweight or Obese Individuals: a system review

Objective Irisin is a newly identified myokine, which is mainly secreted by skeleton muscle, adipose and cerebellar. It is shown to be related to some physiology process. The aim of this study is to evaluate the influence of exercise on circulating irisin concentrations in overweight or obese individuals Methods Searches were performed on nine online electronic databases including PubMed, EMbase, The Cochrane Library, web of science, Ebsco, CNKI, VIP, CBM and Wan-Fang Data databases. The search items were irisin, fibronectin type Ⅲ domain-containing protein 5, FNDC5, exercise, training, physical activity, obesity, overweight, obese, body mass index, BMI, adiposity and fat. Randomized controlled trials (RCT) or clinical controlled trials about the effect of exercise on circulating irisin concentrations in overweight or obese individuals in English or Chinese were eligible for the study. The trials compare exercise intervention with no intervention, or combined interventions of exercise and other with other intervention(s), and the exercise intervention is not one acute time. Besides, the trial objects belong to overweight or obese regardless of the judgement’s indicator. According to the criteria, the data extracted by two research independently. If there was disagreement, discussion between all the authors were used to settle. The risk of bias among the included studies was assessed by the Cochrane Collaboration Risk-of-Bias tool, which consists of seven domains and each one was judged to ‘unclear risk’ ‘low risk’, or ‘high risk’ following the recommendations detailed of the Cochrane handbook. Lastly an analysis about the final studies was done.  Results The search identified a total of 855 possible articles. Of those, 364 were removed as duplicates, and the remaining 491 were screened for the titles and abstracts. The full-texts of 56 trials were retrieved to assess for eligibility. After the evaluation, four articles of RCTs were retained for the final system review from the year of 2015 to 2017, producing 6 study estimates. The assessments class of methods quantality of them are A. All the research subjects are more than 18 years old, and in one study subjects are men, men and women in two, women in three. The types of exercise intervention are dissimilar, such as strengthen or endurance exercise (including high intensity interval training, HIIT). In the duration of exercise, three studies are 8 weeks, and two for 12 weeks, one for 24 weeks. In circulating irisin, the detection methods of all is enzyme-linked immunosorbent assay, and three are in plasm, three in serum. Furthermore, the concentration unit in five studies is ng/ml, and one is µg/ml. Bonefate suggests that aerobic exercise with the frequency of 3 times per week for 24 week maintains plasma FNDC5/irisin of middle-age obese men, same as 8 weeks aerobic exercise for overweight/obese adults by Kim, but three is an opposite result from Wu, which proved that aerobic exercise of twelve weeks ascends serum irisin of young obese women. HIIT of eight or twelve weeks ascends serum irisin in sedentary obese women or young obese women according to Tofighi or Wu suggestion. Moreover, resistance exercise of 8 weeks significantly increases plasma irisin of overweight/obese adults From Kim’s study. Conclusions The study about effect of exercise on circulating irisin levels in overweight or obese individuals is not sufficient to come to a positive result, although the quality assessments of current evidences are high. Basing on the available literatures, exercise can maintain or improve circulating irisin levels in overweight or obese individuals. The effect needs to be illustrated by further RCTs with large sample size. &nbsp

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane 1 protein to reduce ER-localised proteins

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis ensuring correct protein folding and facilitating the degradation of misfolded ER proteins

Rates and kinematics of active shortening along the eastern Qilian Shan, China, inferred from deformed fluvial terraces

In the eastern Qilian Shan, a flight of fluvial terraces developed along the Jinta River valley are deformed across the Nanying anticline. Four individual fluvial terraces are preserved at different elevations above the river, and higher terrace treads are draped by systematically thicker aeolian loess. Optically stimulated luminescence dating of deposits at the base of the loess provides constraints on the timing of surface abandonment; terraces were abandoned at 69 ± 4 ka B.P. (T4), 57 ± 4 ka B.P. (T3), and between 34 ± 3 ka B.P. (T2), respectively. Differential GPS measurement of the terrace profile across the anticline allows reconstruction of subsurface fault geometry; we model terrace deformation above a listric thrust fault with a tip line at 2.2 ± 0.1 km depth and whose dip shallows systematically to 23 ± 3° at depth of 5.8 ± 1.1 km. Combining terrace ages with this model of fault geometry, we estimate a shortening rate of 0.8 ± 0.2 mm/a across the Nanying fold and a shortening rate of ~0.1 mm/a across the mountain front fault since ~70 ka B.P. This rate suggests that the frontal fault system along the eastern Qilian Shan accomplishes crustal shortening at rates of approximately 0.9 ± 0.3 mm/a during late Pleistocene time.This is the publisher’s final pdf. The article is copyrighted by American Geophysical Union and published by John Wiley & Sons, Inc. It can be found at: http://agupubs.onlinelibrary.wiley.com/agu/journal/10.1002/%28ISSN%291944-9194