Acute Resistance Exercise Induces Sestrin2 Phosphorylation and p62 Dephosphorylation in Human Skeletal Muscle

Sestrins (1, 2, 3) are a family of stress-inducible proteins capable of attenuating oxidative stress, regulating metabolism, and stimulating autophagy. Sequestosome1 (p62) is also a stress-inducible multifunctional protein acting as a signaling hub for oxidative stress and selective autophagy. It is unclear whether Sestrin and p62Ser403 are regulated acutely or chronically by resistance exercise (RE) or training (RT) in human skeletal muscle. Therefore, the acute and chronic effects of RE on Sestrin and p62 in human skeletal muscle were examined through two studies. In Study 1, nine active men (22.1 ± 2.2 years) performed a bout of single-leg strength exercises and muscle biopsies were collected before, 2, 24, and 48 h after exercise. In Study 2, 10 active men (21.3 ± 1.9 years) strength trained for 12 weeks (2 days per week) and biopsies were collected pre- and post-training. Acutely, 2 h postexercise, phosphorylation of p62Ser403 was downregulated, while there was a mobility shift of Sestrin2, indicative of increased phosphorylation. Forty-eight hours postexercise, the protein expression of both Sestrin1 and total p62 increased. Chronic exercise had no impact on the gene or protein expression of Sestrin2/3 or p62, but Sestrin1 protein was upregulated. These findings demonstrated an inverse relationship between Sestrin2 and p62 phosphorylation after a single bout of RE, indicating they are transiently regulated. Contrarily, 12 weeks of RT increased protein expression of Sestrin1, suggesting that despite the strong sequence homology of the Sestrin family, they are differentially regulated in response to acute RE and chronic RT

Prediction and Verification of the Major Ingredients and Molecular Targets of Tripterygii Radix Against Rheumatoid Arthritis

Tripterygii Radix exhibits good clinical efficacy and safety in rheumatoid arthritis (RA) patients, but its effective components and mechanism of action are still unclear. The purpose of this study was to explore and verify the major ingredients and molecular targets of Tripterygii Radix in RA using drug-compounds-biotargets-diseases network and protein-protein interaction (PPI) network analyses. The processes and pathways were derived from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The most important compounds and biotargets were determined based on the degree values. RA fibroblast-like synoviocytes (RA-FLS) were separated from RA patients and identified by hematoxylin and eosin (HE) staining and immunohistochemistry. The purity of RA-FLS was acquired by flow cytometry marked with CD90 or VCAM-1. RA-FLS were subjected to control, dimethyl sulfoxide (control), kaempferol, or lenalidomide treatment. Cell migration was evaluated by the transwell assay. The relative expression of biotarget proteins and cytokines was analyzed by western blotting and flow cytometry. In total, 144 chemical components were identified from Tripterygii Radix; kaempferol was the most active ingredient among 33 other components. Fourteen proteins were found to be affected in RA from 285 common biotargets. The tumor necrosis factor (TNF) signaling pathway was predicted to be one of the most latent treatment pathways. Migration of RA-FLS was inhibited and the expression of protein kinase B (AKT1), JUN, caspase 3 (CASP3), TNF receptor 1 and 2 (TNFR1 and TNFR2), interleukin-6 (IL-6), and TNF-α was significantly affected by kaempferol. Thus, this study confirmed kaempferol as the effective component of Tripterygii Radix against RA-FLS and TNF signaling pathway and its involvement in the regulation of AKT1, JUN, CASP3, TNFR1, TNFR2, IL-6, and TNF-α expression

MicroRNAs in muscle: Characterizing the powerlifter phenotype

Powerlifters are the epitome of muscular adaptation and are able to generate extreme forces. The molecular mechanisms underpinning the significant capacity for force generation and hypertrophy are not fully elucidated. MicroRNAs (miRs) are short non-coding RNA sequences that control gene expression via promotion of transcript breakdown and/or translational inhibition. Differences in basal miR expression may partially account for phenotypic differences in muscle mass and function between powerlifters and untrained age-matched controls. Muscle biopsies were obtained from m. vastus lateralis of 15 national level powerlifters (25.1 ± 5.8 years) and 13 untrained controls (24.1 ± 2.0 years). The powerlifters were stronger than the controls (isokinetic knee extension at 60°/s: 307.8 ± 51.6 Nm vs. 211.9 ± 41.9 Nm, respectively P < 0.001), and also had larger muscle fibers (type I CSA 9,122 ± 1,238 vs. 4,511 ± 798 μm2 p < 0.001 and type II CSA 11,100 ± 1,656 vs. 5,468 ± 1,477 μm2 p < 0.001). Of the 17 miRs species analyzed, 12 were differently expressed (p < 0.05) between groups with 7 being more abundant in powerlifters and five having lower expression. Established transcriptionally regulated miR downstream gene targets involved in muscle mass regulation, including myostatin and MyoD, were also differentially expressed between groups. Correlation analysis demonstrates the abundance of eight miRs was correlated to phenotype including peak strength, fiber size, satellite cell abundance, and fiber type regardless of grouping. The unique miR expression profiles between groups allow for categorization of individuals as either powerlifter or healthy controls based on a five miR signature (miR-126, -23b, -16, -23a, -15a) with considerable accuracy (100%). Thus, this unique miR expression may be important to the characterization of the powerlifter phenotype.publishedVersionnivå

Effect of probe characteristics on the subtractive hybridization efficiency of human genomic DNA

<p>Abstract</p> <p>Background</p> <p>The detection sensitivity of low abundance pathogenic species by polymerase chain reaction (PCR) can be significantly enhanced by removing host nucleic acids. This selective removal can be performed using a magnetic bead-based solid phase with covalently immobilized capture probes. One of the requirements to attain efficient host background nucleic acids subtraction is the capture probe characteristics.</p> <p>Findings</p> <p>In this study we investigate how various capture probe characteristics influence the subtraction efficiency. While the primary focus of this report is the impact of probe length, we also studied the impact of probe conformation as well as the amount of capture probe attached to the solid phase. The probes were immobilized on magnetic microbeads functionalized with a phosphorous dendrimer. The subtraction efficiency was assessed by quantitative real time PCR using a single-step capture protocol and genomic DNA as target. Our results indicate that short probes (100 to 200 bp) exhibit the best subtraction efficiency. Additionally, higher subtraction efficiencies with these probes were obtained as the amount of probe immobilized on the solid phase decreased. Under optimal probes condition, our protocol showed a 90 - 95% subtraction efficiency of human genomic DNA.</p> <p>Conclusions</p> <p>The characteristics of the capture probe are important for the design of efficient solid phases. The length, conformation and abundance of the probes determine the capture efficiency of the solid phase.</p

Influence of Traffic Activity on Heavy Metal Concentrations of Roadside Farmland Soil in Mountainous Areas

Emission of heavy metals from traffic activities is an important pollution source to roadside farmland ecosystems. However, little previous research has been conducted to investigate heavy metal concentrations of roadside farmland soil in mountainous areas. Owing to more complex roadside environments and more intense driving conditions on mountainous highways, heavy metal accumulation and distribution patterns in farmland soil due to traffic activity could be different from those on plain highways. In this study, design factors including altitude, roadside distance, terrain, and tree protection were considered to analyze their influences on Cu, Zn, Cd, and Pb concentrations in farmland soils along a mountain highway around Kathmandu, Nepal. On average, the concentrations of Cu, Zn, Cd, and Pb at the sampling sites are lower than the tolerable levels. Correspondingly, pollution index analysis does not show serious roadside pollution owing to traffic emissions either. However, some maximum Zn, Cd, and Pb concentrations are close to or higher than the tolerable level, indicating that although average accumulations of heavy metals pose no hazard in the region, some spots with peak concentrations may be severely polluted. The correlation analysis indicates that either Cu or Cd content is found to be significantly correlated with Zn and Pb content while there is no significant correlation between Cu and Cd. The pattern can be reasonably explained by the vehicular heavy metal emission mechanisms, which proves the heavy metals’ homology of the traffic pollution source. Furthermore, the independent factors show complex interaction effects on heavy metal concentrations in the mountainous roadside soil, which indicate quite a different distribution pattern from previous studies focusing on urban roadside environments. It is found that the Pb concentration in the downgrade roadside soil is significantly lower than that in the upgrade soil while the Zn concentration in the downgrade roadside soil is marginally higher than in the upgrade soil; and the concentrations of Cu and Pb in the roadside soils with tree protection are significantly lower than those without tree protection. However, the attenuation pattern of heavy metal concentrations as a function of roadside distance within a 100 m range cannot be identified consistently

Online calculator for individual affiliation to a major population group

Because of their sensitivity and high level of discrimination, short tandem repeat (STR) maker systems are currently the method of choice in routine forensic casework and data banking, usually in multiplexes up to 15–17 loci. Constraints related to sample amount and quality, frequently encountered in forensic casework, will not allow to change this picture in the near future, notwithstanding the technological developments. In this study, we present a free online calculator named PopAffiliator (http://cracs.fc.up.pt/popaffiliator) for individual population affiliation in the three main population groups, Eurasian, East Asian and sub-Saharan African, based on genotype profiles for the common set of STRs used in forensics. This calculator performs affiliation based on a model constructed using machine learning techniques. The model was constructed using a data set of approximately fifteen thousand individuals collected for this work. The accuracy of individual population affiliation is approximately 86%, showing that the common set of STRs routinely used in forensics provide a considerable amount of information for population assignment, in addition to being excellent for individual identification

CRYPTOCHROMES promote daily protein homeostasis.

The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild-type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild-type controls. Most strikingly, the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating large-scale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to maintain protein and osmotic homeostasis against daily perturbation