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Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phosphomimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis. [ABSTRACT FROM AUTHOR]

Adenosine monophosphate‐activated protein kinase (AMPK) plays pivotal roles in metabolic diseases including type 2 diabetes. However, the specific role of AMPK for orthodontic tooth movement in type 2 diabetes is unclear. In this study, a diabetic rat model was established through dietary manipulation and streptozocin injection. Examinations were conducted to select qualified type 2 diabetic rats. Then, an orthodontic device was applied to these rats for 0, 3, 7, or 14 days. The distance of orthodontic tooth movement and parameters of alveolar bone were analyzed by micro‐computed tomography. Periodontal osteoclastic activity, inflammatory status, and AMPK activity were measured via histological analyses. Next, we repeated the establishment of diabetic rats to investigate whether change of AMPK activity was associated with orthodontic tooth movement under type 2 diabetes. The results showed that diabetic rats exhibited an exacerbated alveolar bone resorption, overactive inflammation, and decreased periodontal AMPK activity during orthodontic tooth movement. Injection of the AMPK agonist alleviated type 2 diabetes‐induced periodontal inflammation and alveolar bone resorption, thus normalizing distance of orthodontic tooth movement. Our study indicates that type 2 diabetes decreases periodontal AMPK activity, leading to excessive inflammation elevating osteoclast formation and alveolar bone resorption, which could be reversed by AMPK activation. [ABSTRACT FROM AUTHOR]

Background/Aim: Rho kinase is a downstream effector of Rho GTPase that is known to regulate various pathological processes. The aim of this study was to evaluate the regulation of Rho kinase activity in leukocytes in patients with ischemia/reperfusion (I/R) injury. Patients and Methods: We investigated 38 patients with acute ST-segment elevation myocardial infarction (STEMI), 26 patients with atherosclerosis (AS) and 22 normal subjects. All patients underwent coronary angiography (CAG) and all STEMI patients received primary percutaneous coronary intervention (PPCI) of the left anterior descending artery (LAD) within 12 h after chest pain on-set. Blood samples for leukocyte Rho kinase activity were obtained before CAG and 3 and 24 hours after CAG/PCI. Results: Rho kinase activity increased in the I/R and AS groups. Compared with the AS group, Rho kinase activity was significantly higher in peripheral blood leukocytes in STEMI/PPCI. Furthermore, there was no correlation between changes in Rho kinase activity and changes in high-sensitivity troponin I (hs-TnI) and C-reactive protein (CRP). There was a negative correlation between Rho kinase activity and IL-6. Conclusion: Rho kinase is involved in the pathogenesis of heart I/R injury in patients. Inhibition of Rho kinase may be an additional therapeutic intervention for the treatment of I/R. [ABSTRACT FROM AUTHOR]

Background: Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance.Methods: We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm  with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4-11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors.Results: Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4-11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4-11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks.Conclusions: We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples. [ABSTRACT FROM AUTHOR]

Background: Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T.Methods: From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity.Results: In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype.Conclusion: Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age. [ABSTRACT FROM AUTHOR]

Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations inPTENfrequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygousPTENdeficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizesPTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation inPTEN-deficient lipoma cells. Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis.PTENexpression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced inPTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primaryPTENwild-type preadipocytes were less affected compared toPTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes. [ABSTRACT FROM PUBLISHER]

Objective. Aberrant rho kinase (ROCK) activity is implicated in the pathogenesis of several vascular diseases and is associated with Th17 differentiation. Th17 immune response is recognized in the pathogenesis of GCA. The aim of this study was to assess ROCK activity in GCA.Methods. All patients who underwent temporal artery biopsy (TAB) at a tertiary care centre over 5 years were identified and charts reviewed. Subjects were categorized into three groups: TAB-positive GCA, TAB-negative GCA and age- and sex-matched controls. TABs were stained for phosphorylated ezrin/radixin/moesin (pERM), a surrogate of ROCK activity, and reviewed by a pathologist blinded to clinical status. Three areas were scored for staining intensity on a scale of 0–2, with a maximum possible score of 6.Results. Nineteen subjects with TAB-positive GCA, 17 with TAB-negative GCA and 18 controls were analysed. Compared with controls, GCA subjects with either positive or negative TABs had significantly higher pERM intensity scores (P = 0.0109). Adjusting for diabetes, hypertension, prednisone and statin use, GCA subjects still had higher pERM scores [odds ratio 7.3 (95% CI 1.9, 25.9), P = 0.0046]. The high pERM score had a sensitivity of 90% and a negative predictive value of 91% for the diagnosis of GCA in those with a negative TAB, compared with 51% sensitivity for histopathology alone.Conclusion. Subjects with GCA had more intense pERM staining in TAB specimens compared with age- and sex-matched controls, regardless of whether TAB was positive or negative by routine histopathology, suggesting increased ROCK activity in GCA. The ROCK pathway warrants further investigation in GCA, as it may have diagnostic significance in enhancing the sensitivity of TAB. [ABSTRACT FROM PUBLISHER]

Background: Spermatozoa are a group of cells that consume adenosine triphosphate (ATP) rapidly. Creatine kinase (CK), produced by creatine phosphate, is an energy reservoir for the rapid buffering and regeneration of ATP and can play an important role in sperm motility. Therefore, this study investigates the effects of cigarette smoking on human sperm CK activity in males who smoke. Materials and Methods: In this case - control study, we obtained semen samples from male smokers (n=64) and nonsmokers (n=83). Smokers were categorized as light, moderate, or heavy smokers according to the daily number of cigarettes smoked and the number of years they have smoked. Data were analyzed by the independent t test and Pearson's analysis. Results: This investigation showed significantly lower sperm CK activity and movement in male smokers compared to nonsmokers. In addition, it was demonstrated that cigarette smoking had a dose-dependent effect on these parameters. There was a positive relation, although not significant, between sperm CK activity and its motility in male smokers. Conclusion: Smoking, by diminishing sperm CK activity, may potentially impair sperm energy homeostasis and have an association with damage to sperm motility. This effect can be an important mechanism that may cause infertility in male smokers. However, further research is necessary to elucidate the underlying mechanism of sperm motility damage caused by cigarette smoking. [ABSTRACT FROM AUTHOR]

Objective Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP-activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1β and TNFα. Methods Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr [ABSTRACT FROM AUTHOR]

Objective Anti-signal recognition particle (anti-SRP) autoantibodies are associated with severe acquired necrotizing myopathies. The role of these autoantibodies remains elusive, and the evolution of anti-SRP levels over time is unknown. In this study, we developed an addressable laser bead immunoassay (ALBIA) technique to investigate a correlation between anti-SRP levels, serum creatine kinase (CK) levels, and muscle strength in patients with necrotizing myopathy. Methods The diagnostic value of the ALBIA assay was determined by comparing serum levels of anti-SRP autoantibodies in 31 anti-SRP immunodot-positive patients to those in 190 healthy blood donors and 199 control patients with different inflammatory/autoimmune conditions or polyclonal hypergammaglobulinemia. Among the 31 anti-SRP-positive patients, serum samples from 8 patients were monitored over time for levels of anti-SRP autoantibodies and levels of CK (determined at least 3 times, consecutively, over a mean followup period of 783 days). The relationship between levels of anti-SRP autoantibodies and levels of CK was tested using a linear mixed model. Results The assay yielded positive results for anti-SRP in all anti-SRP immunodot-positive serum samples tested, while all control sera tested negative. The 8 anti-SRP-positive patients who were followed up longitudinally were found to have normalized CK levels and improved muscle strength. There was a striking correlation between the degree of myolysis, as measured by CK levels, in patients receiving therapy and the anti-SRP54 autoantibody levels in these same patients ( P = 0.002). Conclusion Anti-SRP-positive myositis appears to be one of the few autoimmune diseases in which specific autoantibody levels are correlated with surrogate disease activity markers. These results reveal the usefulness of monitoring anti-SRP autoantibody levels in patients receiving therapy, and may also suggest a possible pathogenic role for anti-SRP autoantibodies in the necrotizing myopathies. [ABSTRACT FROM AUTHOR]

Purpose: The authors investigated whether far-infrared radiation (FIR) lamp therapy would reduce muscle damage and enhance recovery from multiple soccer-match-related running activities. Methods: Twenty-four elite female soccer players (20–24 y) were assigned into a FIR or a sham treatment group (n = 12/group). They performed a daily 90-minute Loughborough Intermittent Shuttle Test (LIST) for 6 consecutive days. Maximal voluntary contraction torque of the knee extensors (KEs) and flexors, muscle soreness, plasma creatine kinase activity, countermovement jump, and several other performance measures (eg, 30-m dash, Yo-Yo Intermittent Recovery Test Level 1) were taken before the first LIST, 1 hour after each LIST, and 24, 48, 72, 96, and 120 hours after the last LIST. All participants received a 30-minute FIR or sham treatment on KEs and knee flexors, respectively, at 2 hour after each LIST and 25, 49, 73, and 97 hours after the last LIST. Results: All measures changed significantly (P <.05) at 1 hour after the first LIST without difference (P >.05) between groups. Maximal voluntary contraction torque (eg, the largest decrease of KE for FIR: 13% [4%], sham: 25% [5%]), countermovement jump height (4% [3%] vs 14% [4%]), and other performance measures (eg, Yo-Yo Intermittent Recovery Test: 11% [5%] vs 26% [5%]) decreased less, and peak muscle soreness (eg, KE: 26 [9] vs 51 [18] mm) and plasma creatine kinase activity (172 [32] vs 1289 [610] IU/L) were smaller for the FIR than for the sham group (P <.05), and they returned to the baseline earlier (P <.05) for the FIR group. Conclusions: These results suggest that the FIR therapy provided potent effects on reducing accumulated muscle damage and enhancing recovery. [ABSTRACT FROM AUTHOR]

Background: Huashi Baidu Formula (HBF) is a clinical formula known for its efficacy against coronavirus disease 2019 (COVID-19). HBF may reduce the number of patients with abnormal serum creatinine while improving respiratory symptoms, suggesting that this formula may have potential for treating acute kidney injury (AKI). However, the protective effect of HBF on AKI has not been definitively confirmed, and the mechanism remains unclear. Therefore, the present study explored the renoprotective effects and molecular mechanisms of HBF and screened for its active ingredients to identify new potential applications of renoprotection by HBF. Methods: The present study first assessed the protective effects of HBF on AKI in a DOX-induced mouse model. Then, RNA-seq and bioinformatics analyses were used to explore the related pathological processes and potential molecular mechanisms, which were subsequently validated using qRT-PCR and Western blotting. Furthermore, candidate compounds with potential binding affinity to two pivotal targets, sphingosine kinase 1 (SphK1) and plasminogen activator inhibitor-1 (PAI-1), were screened from the 29 constituents present in the blood using Microscale Thermophoresis (MST). Finally, to identify the active ingredients, the candidate components were re-screened using the SphK1 kinase activity detection system or the uPA/PAI-1 substrate colorimetric assay system. Results: In the DOX-induced AKI mouse model, therapeutic administration of HBF significantly reduced the levels of CRE, BUN, TNF-α, IL-1β, IL-6, and UA in plasma and the levels of MDA, T-CHO, and TG in kidney tissue. Additionally, the levels of TP and Alb in plasma and SOD and CAT in the kidney tissue were significantly increased. Histopathological assessment revealed that HBF reduced tubular vacuolation, renal interstitial inflammatory cell infiltration, tubular atrophy, and positive staining of renal interstitial collagen. RNA-seq and bioinformatics analyses showed that oxidative stress, the immune-inflammatory response, and extracellular matrix (ECM) formation could be the pathological processes that HBF targets to exerts its renoprotective effects. Furthermore, HBF regulated the APJ/SPHK1/NF-κB and APJ/PAI-1/TGFβ signaling axes and reduced the phosphorylation levels of NF-κB p65 and SMAD2 and the expression of cytokines and the ECM downstream of the axis. Finally, six SphK1 inhibitors (paeoniflorin, astragalin, emodin, glycyrrhisoflavone, quercetin, and liquiritigenin) and three PAI-1 inhibitors (glycyrrhisoflavone, licochalcone B, and isoliquiritigenin) were identified as potentially active ingredients in HBF. Conclusion: In brief, our investigation underscores the renoprotective effect of HBF in a DOX-induced AKI model mice, elucidating its mechanisms through distinct pathological processes and identifying key bioactive compounds. These findings offer new insights for broadening the clinical applications of HBF and unravelling its molecular mode of action. [ABSTRACT FROM AUTHOR]

This study aimed to compare the acute inflammatory response following high-intensity eccentric exercise between resistance-trained young and master athletes with similar performance levels. Resistance-trained young (n=8; 22±2 years) and master (n=8; 52±4 years) male athletes of a similar performance level performed a standardized high-intensity eccentric squat exercise protocol (10 sets of half-squats at 70% of 1-repetition maximum). The serum concentration of 20 biomarkers related to tissue damage, inflammation, remodeling, and repair was measured at baseline, immediately after exercise, and over a 72 h recovery period. Both groups experienced similar muscle damage as evidenced by a comparable increase in creatine kinase activity 24 h after exercise (p<0.001). Interleukin-6 (p=0.009) and growth hormone (p<0.001) increased immediately post-exercise in both groups. Monocyte chemoattractant protein-1 increased immediately post-exercise only in young athletes (p=0.003) and then decreased 24 h later. There were no significant differences for the remaining variables, including cell markers related to neutrophil/macrophage activation or pro/anti-inflammatory cytokines. Resistance-trained young and master athletes, matched for performance level, showed an overall similar inflammatory response to eccentric exercise, possibly reflecting regulatory mechanisms or immunological adaptations to chronic stimulation in master athletes. [ABSTRACT FROM AUTHOR]

Flavonoids reduce cardiovascular disease risk through anti-inflammatory, anti-coagulant and anti-platelet actions. One key flavonoid inhibitory mechanism is blocking kinase activity that drives these processes. Flavonoids attenuate activities of kinases including phosphoinositide-3-kinase, Fyn, Lyn, Src, Syk, PKC, PIM1/2, ERK, JNK and PKA. X-ray crystallographic analyses of kinase-flavonoid complexes show that flavonoid ring systems and their hydroxyl substitutions are important structural features for their binding to kinases. A clearer understanding of structural interactions of flavonoids with kinases is necessary to allow construction of more potent and selective counterparts. We examined flavonoid (quercetin, apigenin and catechin) interactions with Src family kinases (Lyn, Fyn and Hck) applying the Sybyl docking algorithm and GRID. A homology model (Lyn) was used in our analyses to demonstrate that high-quality predicted kinase structures are suitable for flavonoid computational studies. Our docking results revealed potential hydrogen bond contacts between flavonoid hydroxyls and kinase catalytic site residues. Identification of plausible contacts indicated that quercetin formed the most energetically stable interactions, apigenin lacked hydroxyl groups necessary for important contacts and the non-planar structure of catechin could not support predicted hydrogen bonding patterns. GRID analysis using a hydroxyl functional group supported docking results. Based on these findings, we predicted that quercetin would inhibit activities of Src family kinases with greater potency than apigenin and catechin. We validated this prediction using in vitro kinase assays. We conclude that our study can be used as a basis to construct virtual flavonoid interaction libraries to guide drug discovery using these compounds as molecular templates. [ABSTRACT FROM AUTHOR]

Kinase suppressor of Ras-1 (KSR1) is a recently identified member of the EGFR-Ras-Raf-1-MAPK signaling pathway. A new study demonstrates that KSR1 protects intestinal epithelium from TNF-alpha-induced apoptosis, abrogating inflammatory bowel disease (IBD). Since its discovery, there has been disagreement as to whether KSR1 possesses intrinsic kinase activity. Using transgenic mouse models and genetically modified mouse colon epithelial cells, Polk and coworkers show that the kinase activity of KSR1 is off in normal colon epithelial cells, becoming activated only at the onset of IBD. They also provide strong evidence that KSR1 kinase activity is essential for anti-apoptotic protection of the intestinal epithelium. These new data in support of KSR1 as a kinase highlight an ongoing debate as to whether KSR1 does indeed serve as a specific kinase in transphosphorylating and transactivating c-Raf-1 toward MEK1. [ABSTRACT FROM AUTHOR]

Introduction: Vascular smooth muscle cells are important in intimal hyperplasia. Thrombospondin-1 is a matricellular protein involved in the vascular injury response. Statins are cholesterol lowering drugs that have beneficial cardiovascular effects. Statis have been shown to inhibit smooth muscle migration through the mevalonate pathway. This effect is thought to be mediated by small G protein Ras and Rho turnover which requires many hours. While many patients undergoing treatment for vascular disease are on statins, many are not. Thus immediate pretreatment with statins before surgery may be beneficial. We hypothesized that statins have effects independent of the mevalonate pathway and thus have an immediate effect. Methods: Human vascular smooth muscle cells were pretreated for 20 h (long-term) or 20 min (short-term) with fluvastatin, or mevalonolactone plus fluvastatin. Thrombospondin-1-induced migration, activation of p42/p44 extracellular signal-regulated kinase, c-Src, focal adhesion kinase and PI3 kinase was determined. The effect of fluvastatin on thrombospondin-1-induced expression of THBS1, FOS, HAS2 and TGFB2 was examined. Results: Both treatments inhibited thrombospondin-1-induced chemotaxis back to the control group. Mevalonolactone reversed the long-term statin effect by increasing migration but had no effect on the short-term statin response. p42/p44 extracellular signal-regulated kinase was activated by thrombospondin-1 and both treatments augmented activation. Neither treatment affected c-Src activity, but both inhibited focal adhesion kinase and PI3 kinase activity. Only long-term statin treatment inhibited THBS1 expression while both treatments inhibited FOS and TGFB2 expression. Neither treatment affected HAS2. FOS knockdown inhibited thrombospondin-1-induced HAS2 but not TGFβ2 gene expression. Conclusion: Long-term fluvastatin inhibited thrombospondin-1-induced chemotaxis through the mevalonate pathway while short-term fluvastatin inhibited chemotaxis through an alternate mechanism. Short-term stains have immediate effects independent of the mevalonate pathway. Acute local treatment with statins followed by longer term therapy may limit the vascular response to injury. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of a 3‐week immobilization (IM) on muscle damage induced by maximal eccentric exercise (MaxEC) to test the hypothesis that the IM would make muscles prone to muscle damage. Young healthy sedentary men were pseudo‐randomly assigned to IM or control group (n = 12/group). Non‐dominant arms of the IM group participants were immobilized at 90° elbow flexion by a cast for 21 days. All participants performed MaxEC consisting of five sets of six elbow flexor contractions by lowering a dumbbell set at 100% of pre‐exercise maximal voluntary isometric contraction (MVC) strength of the non‐dominant arm. This was performed at 2 days after the cast removal for the IM group. MVC torque, range of motion (ROM), muscle thickness (MT), muscle hardness, position sense (PS), and joint reaction angle (JRA) of the elbow flexors were measured at baseline, post‐immobilization, and before, immediately after, and one to 5 days after MaxEC. The IM decreased MVC torque (−17 ± 2%), ROM (−2 ± 1%), MT (−7 ± 3%), and JRA (−12 ± 6%), and increased in muscle hardness (20 ± 6%) and PS (11 ± 2%) (p < 0.05). Changes in MVC (e.g., 2 days: −40 ± 5 vs. −30 ± 9%), ROM (2 days: −11 ± 2 vs. −9 ± 3%), muscle soreness (peak: 63 ± 22 vs. 48 ± 14 mm), plasma CK activity (peak: 7820 ± 4011 vs. 4980 ± 1363 IU/L), PS (maximal change: −23 ± 2 vs. −18 ± 3%), and JRA (maximal change: −37 ± 4 vs. −26 ± 3%) after MaxEC were greater (p < 0.05) for the IM than control group. These results supported the hypothesis and showed that the IM made the muscles more vulnerable to muscle damage induced by eccentric exercise. [ABSTRACT FROM AUTHOR]

Simple Summary: Tyrosine kinase inhibitors (TKIs), particularly imatinib, have afforded Chronic Myeloid Leukemia (CML) patients long-term remission. This revolution in leukemia treatment is unfortunately accompanied by the fact that treatment interruption, in most cases, leads to the re-emergence of BCR-ABL-leukemia cells, caused by leukemia stem cells, that are insensitive to TKI treatment. Moreover, TKIs paradoxically induce a quiescent state in leukemia stem and progenitor cells. Current CML studies focus on these quiescent leukemia stem cells in order to reactivate them and restore TKI sensitivity. We show here that under TKI treatment, leukemia stem cells retrieve motility in a reconstituted niche when incubated with SKF96365, a calcium channel inhibitor, and that this effect is dependent on RhoA activation by BCR-ABL independently of its tyrosine kinase activity. Altogether, these results suggest that SKF-96365 or its derived molecules could be interesting compounds for targeting quiescent CML stem and progenitor cells under TKI treatment. Background: A major issue in Chronic Myeloid Leukemia (CML) is the persistence of quiescent leukemia stem cells (LSCs) in the hematopoietic niche under tyrosine kinase inhibitor (TKI) treatment. Results: Here, using CFSE sorting, we show that low-proliferating CD34+ cells from CML patients in 3D co-culture hide under HS27A stromal cells during TKI treatment—a behavior less observed in untreated cells. Under the same conditions, Ba/F3p210 cells lose their spontaneous motility. In CML CD34+ and Ba/F3p210 cells, while Rac1 is completely inhibited by TKI, RhoA remains activated but is unable to signal to ROCK. Co-incubation of Ba/F3p210 cells with TKI, SKF-96365 (a calcium channel inhibitor), and EGF restores myosin II activation and amoeboid motility to levels comparable to untreated cells, sustaining the activation of ROCK. In CFSE+ CD34+ cells containing quiescent leukemic stem cells, co-incubation of TKI with SKF-96365 induced the expulsion of these cells from the HS27A niche. Conclusions: This study underscores the role of RhoA in LSC behavior under TKI treatment and suggests that SKF-96365 could remobilize quiescent CML LSCs through reactivation of the RhoA/ROCK pathway. [ABSTRACT FROM AUTHOR]

Simple Summary: Here, we show that the plant-derived product, sanguinarine, inhibited aerobic glycolysis and oxidative phosphorylation; which resulted in energy alternations and necroptosis of tumor cells. Moreover, we identify the PKM2/β-catenin axis as the main target in sanguinarine treatment against HCC development. Backgrounds: Abnormal metabolism is the hallmark of hepatocellular carcinoma. Targeting energy metabolism has become the major focus of cancer therapy. The natural product, sanguinarine, displays remarkable anti-tumor properties by disturbing energy homeostasis; however, the underlying mechanism has not yet been elucidated. Methods: The anticancer activity of sanguinarine was determined using CCK-8 and colony formation assay. Morphological changes of induced cell death were observed under electron microscopy. Necroptosis and apoptosis related markers were detected using western blotting. PKM2 was identified as the target by transcriptome sequencing. Molecular docking assay was used to evaluate the binding affinity of sanguinarine to the PKM2 molecule. Furthermore, Alb-CreERT2; PKM2loxp/loxp; Rosa26RFP mice was used to construct the model of HCC—through the intervention of sanguinarine in vitro and in vivo—to accurately explore the regulation effect of sanguinarine on cancer energy metabolism. Results: Sanguinarine inhibited tumor proliferation, metastasis and induced two modes of cell death. Molecular docking of sanguinarine with PKM2 showed appreciable binding affinity. PKM2 kinase activity and aerobic glycolysis rate declined, and mitochondrial oxidative phosphorylation was inhibited by sanguinarine application; these changes result in energy deficits and lead to necroptosis. Additionally, sanguinarine treatment prevents the translocation of PKM2 into the nucleus and suppresses the interaction of PKM2 with β-catenin; the transcriptional activity of PKM2/β-catenin signaling and its downstream genes were decreased. Conclusions: Sanguinarine showed remarkable anti-HCC activity via regulating energy metabolism by PKM2/β-catenin signaling. On the basis of these investigations, we propose that sanguinarine might be considered as a promising compound for discovery of anti-HCC drugs. [ABSTRACT FROM AUTHOR]

Objective: To characterize potential mechanisms of fisetin on hepatic insulin resistance (IR) using network pharmacology and in vitro validation. Methods: Putative targets of fisetin were retrieved from the Traditional Chinese Medicine Systems Pharmacology database, whereas the potential genes of hepatic IR were obtained from GeneCards database. A protein–protein interaction (PPI) network was constructed according to the intersection targets of fisetin and hepatic IR using the Venn diagram. The biological functions and potential pathways related to genes were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Cell experiments were also conducted to further verify the mechanism of fisetin on hepatic IR. Results: A total of 118 potential targets from fisetin were associated with hepatic IR. The areas of nodes and corresponding degree values of TP53, AKT1, TNF, IL6, CASP3, CTNNB1, JUN, SRC, epidermal growth factor receptor (EGFR), and HSP90AA1 were larger and could be easily found in the PPI network. Furthermore, GO analysis revealed that these key targets were significantly involved in multiple biological processes that participated in oxidative stress and serine/threonine kinase activity. KEGG enrichment analysis showed that the PI3K/AKT signaling pathway was a significant pathway involved in hepatic IR. Our in vitro results demonstrated that fisetin treatment increased the expressions of EGFR and IRS in HepG2 and L02 cells under normal or IR conditions. Western blot results revealed that p-AKT/AKT levels were significantly up-regulated, suggesting that fisetin was involved in the PI3K/AKT signaling pathway to regulate insulin signaling. Conclusion: We explored the pharmacological actions and the potential molecular mechanism of fisetin in treating hepatic IR from a holistic perspective. Our study lays a theoretical foundation for the development of fisetin for type 2 diabetes. [ABSTRACT FROM AUTHOR]

Objective: Variant ataxia-telangiectasia is caused by mutations that allow some retained ataxia telangiectasia-mutated (ATM) kinase activity. Here, we describe the clinical features of the largest established cohort of individuals with variant ataxia-telangiectasia and explore genotype-phenotype correlations.Methods: Cross-sectional data were collected retrospectively. Patients were classified as variant ataxia-telangiectasia based on retained ATM kinase activity.Results: The study includes 57 individuals. Mean age at assessment was 37.5 years. Most had their first symptoms by age 10 (81%). There was a diagnostic delay of more than 10 years in 68% and more than 20 years in one third of probands. Disease severity was mild in one third of patients, and 43% were still ambulant 20 years after disease onset. Only one third had predominant ataxia, and 18% had a pure extrapyramidal presentation. Individuals with extrapyramidal presentations had milder neurological disease severity. There were no significant respiratory or immunological complications, but 25% of individuals had a history of malignancy. Missense mutations were associated with milder neurological disease severity, but with a higher risk of malignancy, compared to leaky splice site mutations.Interpretation: Individuals with variant ataxia-telangiectasia require malignancy surveillance and tailored management. However, our data suggest the condition may sometimes be mis- or underdiagnosed because of atypical features, including exclusive extrapyramidal symptoms, normal eye movements, and normal alpha-fetoprotein levels in some individuals. Missense mutations are associated with milder neurological presentations, but a particularly high malignancy risk, and it is important for clinicians to be aware of these phenotypes. ANN NEUROL 2019;85:170-180. [ABSTRACT FROM AUTHOR]

Simple Summary: The aim of our study is to understand the signaling mechanisms behind the tumor suppressive function of RIPK4 (receptor-interacting serine/threonine protein kinase 4) in squamous cell carcinomas (SCCs) of skin, head, and neck regions. SCCs are some of the deadliest cancers, with poor survival rate, and understanding molecular mechanisms behind the tumor formation will help us to develop effective therapies. Through gene profiling and targeted CRISPR screening approaches in mouse models of cancer, we identified ELOVL4, (elongation of very-long-chain fatty acid-4) as a downstream target of RIPK4. Previous studies have shown that RIPK4 is a NOTCH target gene and RIPK4 phosphorylates IRF6 (interferon regulatory factor 6). Our data from in vitro and in vivo mouse experiments show that the NOTCH-RIPK4-IRF6-ELOVL4 signaling axis functions as a potent tumor suppressor in SCCs. Receptor-interacting serine/threonine protein kinase 4 (RIPK4) and its kinase substrate the transcription factor interferon regulatory factor 6 (IRF6) play critical roles in the development and maintenance of the epidermis. In addition, ourselves and others have previously shown that RIPK4 is a NOTCH target gene that suppresses the development of cutaneous and head and neck squamous cell carcinomas (HNSCCs). In this study, we used autochthonous mouse models, where the expression of Pik3caH1047R oncogene predisposes the skin and oral cavity to tumor development, and show that not only loss of Ripk4, but also loss of its kinase substrate Irf6, triggers rapid SCC development. In vivo rescue experiments using Ripk4 or a kinase-dead Ripk4 mutant showed that the tumor suppressive function of Ripk4 is dependent on its kinase activity. To elucidate critical mediators of this tumor suppressive pathway, we performed transcriptional profiling of Ripk4-deficient epidermal cells followed by multiplexed in vivo CRISPR screening to identify genes with tumor suppressive capabilities. We show that Elovl4 is a critical Notch-Ripk4-Irf6 downstream target gene, and that Elovl4 loss itself triggers SCC development. Importantly, overexpression of Elovl4 suppressed tumor growth of Ripk4-deficient keratinocytes. Altogether, our work identifies a potent Notch1-Ripk4-Irf6-Elovl4 tumor suppressor axis. [ABSTRACT FROM AUTHOR]

The current study aims to confirm the positive effects of dietary nano-Se on nutrients deposition and muscle fibre formation in grass carp fed with high-fat diet (HFD) before overwintering and to reveal its possible molecular mechanism. The lipid deposition, protein synthesis and muscle fibre formation in grass carp fed with regular diet (RD), HFD or HFD supplemented with nano-Se (0·3 or 0·6 mg/kg) for 60 d were tested. Results show that nano-Se significantly reduced lipid content, dripping loss and fibre diameter (P < 0·05), but increased protein content, post-mortem pH24 h and muscle fibre density (P < 0·05) in muscle of grass carp fed with HFD. Notably, dietary nano-Se decreased lipid deposition in the muscle by regulating amp-activated protein kinase activity and increased protein synthesis and fibre formation in muscle by activating target of rapamycin and myogenic determining factors pathways. In summary, dietary nano-Se can regulate the nutrients deposition and muscle fibre formation in grass carp fed with HFD, which exhibit potential benefit for improving flesh quality of grass carp fed with HFD. [ABSTRACT FROM AUTHOR]

Background: Neuropathic pain (NP) is the most prevalent form of chronic pain resulting from nerve damage or injury. Despite the widespread use of Duhuo Jisheng decoction (DHJSD) in traditional Chinese medicine (TCM) to treat chronic pain, the mechanism underlying its analgesic action remains unclear. Methods: Using network pharmacology, we obtained DHJSD and NP-related target information from public databases to construct protein–protein interactions (PPI) and compound-target networks based on common target genes. These networks were further analyzed using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG). The interaction between molecules was verified through molecular docking using AutoDock Tools software. Additionally, we treated a chronic constriction injury (CCI) rat model with DHJSD and determined the mechanical withdrawal threshold (MWT). We used an enzyme-linked immunosorbent assay kit to determine the levels of inflammatory cytokines. Furthermore, qRT-PCR was employed to analyze ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA expression, and immunofluorescence was used to evaluate changes in microglia. Results: Our screening of compounds and targets identified 252 potential targets of DHJSD associated with NP. PPI analysis, along with GO and KEGG analyses, revealed that the potential mechanism of DHJSD in NP treatment may be related to inflammatory reactions, the IL-17 signaling pathway, MAP kinase activity, and endocrine activity. Based on molecular docking, the core target showed significant affinity for DHJSD's active components. Moreover, DHJSD treatment repaired the CCI-induced inflammatory reaction in the spinal cord while regulating the expression of ACHE, NOS2, MAPK3, PTGS2, AKT1, and PPARG mRNA. Immunofluorescence results indicated that the active components of DHJSD may regulate microglial M1 polarization to improve neuroinflammation, PPARG may have been involved in the process. Conclusion: The multi-component, multi-target, and multi-pathway actions of DHJSD provide new insights into its therapeutic mechanism in NP. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is the most common cancer among women worldwide. Overexpression of the HER2 receptor is associated with a worse prognosis and poorer survival. Consequently, several anti-HER2 therapies have been developed, such as trastuzumab. However, resistance still affects a significant population and is currently a major challenge in clinical oncology. Therefore, this study aims to identify biomarkers to predict disease progression and anticipate the efficacy of therapy to avoid therapeutic failure. We identified deregulated genes in trastuzumab-resistant cells associated with cell adhesion and migration. We demonstrate that combined anti-HER2 therapies are encouraging since low doses exhibit a synergism interaction and efficiently inhibit cell adhesion and migration critical process in cancer metastasis. We found deregulated proteins in resistant cells that may be potential biomarkers of response to therapy and may be involved in therapy resistance, useful to predict survival and response to trastuzumab therapy in HER2-positive breast cancer patients. These findings are promising for personalized breast cancer management to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies. Breast cancer (BC) is the most common malignancy among women worldwide. Around 15–25% of BC overexpress the human epidermal growth factor receptor 2 (HER2), which is associated with a worse prognosis and shortened disease-free survival. Therefore, anti-HER2 therapies have been developed, such as monoclonal antibodies (trastuzumab, Tz), antibody–drug conjugates (ado-trastuzumab emtansine, T-DM1), and pharmacological inhibitors of tyrosine kinase activity (lapatinib, Lp). Although Tz, the standard treatment, has significantly improved the prognosis of patients, resistance still affects a significant population of women and is currently a major challenge in clinical oncology. Therefore, this study aims to identify potential biomarkers to predict disease progression (prognostic markers) and the efficacy of Tz treatment (predictive markers) in patients with HER2+ BC. We hypothesize that proteins involved in cell motility are implicated in Tz-resistance. We aim to identify alterations in Tz-resistant cells to guide more efficient oncologic decisions. By bioinformatics, we selected candidate proteins and determined how their expression, localization, and the process they modulate were affected by anti-HER2 treatments. Next, using HER2+ BC patients' data, we assessed these proteins as prognostic and predictive biomarkers. Finally, using Tz-resistant cells, we evaluated their roles in Tz response. We identified deregulated genes associated with cell motility in Tz/T-DM1-resistant vs. -sensitive cells. We showed that Tz, T-DM1, and Lp decrease cell viability, and their effect is enhanced in combinations. We determined synergism between Tz/T-DM1 and Lp, making possible a dose reduction of each drug to achieve the same therapeutic effect. We found that combinations (Tz/T-DM1 + Lp) efficiently inhibit cell adhesion and migration. Furthermore, we demonstrated the induction of FAK nuclear and cortactin peri-nuclear localization after T-DM1, Lp, and Tz/T-DM1 + Lp treatments. In parallel, we observed that combined treatments downregulate proteins essential for metastatic dissemination, such as SRC, FAK, and paxillin. We found that low vinculin (VCL) and cortactin (CTTN) mRNA expression predicts favorable survival rates and has diagnostic value to discriminate between Tz-sensible and Tz-resistant HER2+ BC patients. Finally, we confirmed that vinculin and cortactin are overexpressed in Tz-resistance cells, SKBR3-RTz. Moreover, we found that Tz plus FAK/paxillin/cortactin-silencing reduced cell adhesion/migration capacity in Tz-sensitive and -resistant cells. In conclusion, we demonstrate that combined therapies are encouraging since low doses of Tz/T-DM1 + Lp inhibit metastatic processes by downregulating critical protein expression and affecting its subcellular localization. We propose that vinculin and cortactin might contribute to Tz-sensibility/resistance in BC cells. Finally, we identify potential prognostic and predictive biomarkers that are promising for personalized BC management that would allow efficient patient selection in order to mitigate resistance and maximize the safety and efficacy of anti-HER2 therapies. [ABSTRACT FROM AUTHOR]

The extracts from Psoralea corylifolia Linn. (P. corylifolia) seeds have been shown to display antitumor activity. To date, the prospects of this plant and its active compounds in the treatment of non-small-cell lung cancer (NSCLC) have not been thoroughly studied. In this study, we identified a novel psorachromene compound that displays selective cytotoxic effects on all NSCLC cells tested, including NSCLC cells harboring epidermal growth factor receptor (EGFR) activation mutants (H1975L858R/T790M and H1975-MS35L858R/T790M/C797S ). Psorachromene induces G1 arrest in NSCLC cells harboring wild-type EGFR but induces apoptosis in NSCLC cells harboring activating EGFR mutations. Psorachromene inhibits activated EGFR signaling and kinase activity and suppresses tumor growth of implanted H1975-MS35L858R/T790M/C797S cells in nude mice. Molecular docking analysis revealed that psorachromene could form stronger bonds with mutant EGFR than wild-type EGFR, which might account for the greater cytotoxic effects observed in NSCLC cells harboring activating EGFR mutations (H1975 and H1975-MS35) than wild-type EGFR (A549). In conclusion, it is suggested that psorachromene is an attractive agent to be further explored for its use in the treatment of NSCLC patients harboring EGFR L858R/T790M/C797S. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to examine the recovery kinetics (i.e., time-dependent changes) of performance-related variables between two 120-min male football games performed 3 d apart with and without carbohydrate supplementation. Methods: Twenty male players (20 ± 1 yr; body fat, 14.9% ± 5.1%; maximal oxygen consumption, 59.4 ± 3.7 mL⋅kg-1⋅min-1) participated in two 120-min football games (G1, G2) according to a randomized, two-trial, repeated-measures, crossover, double-blind design. Participants received carbohydrate/placebo supplements during recovery between games. Field activity was monitored during the games. Performance testing and blood sampling were performed before and at 90 and 120 min of each game. Muscle biopsies were collected at baseline and at 90 and 120 min of G1 and pre-G2. Results: Compared with G1, G2 was associated with reduced total distance (10,870 vs 10,685 m during 90 min and 3327 vs 3089 m during extra 30 min; P = 0.007-0.038), average (6.7 vs 6.2 km/h during extra 30-min game-play; P = 0.007) and maximal speed (32.2 vs 30.2 km/h during 90 min and 29.0 vs 27.9 km/h during extra 30 min; P < 0.05), accelerations/decelerations (P < 0.05), and mean heart rate (P < 0.05). Repeated sprint ability (P < 0.001), jumping (P < 0.05), and strength (P < 0.001) performance were compromised before and during G2. Muscle glycogen was not restored at G2 baseline (P = 0.005). Extended game-play reduced lymphocyte, erythrocyte counts, hematocrit, hemoglobin, reduced glutathione (P < 0.05) and increased delayed onset of muscle soreness, creatine kinase activity, blood glycerol, ammonia, and protein carbonyls (P < 0.05) before and during G2. Pax7+ (P = 0.004) and MyoD+ cells (P = 0.019) increased at baseline G2. Carbohydrate supplementation restored performance and glycogen, reduced glycerol and delayed onset of muscle soreness responses, and increased leukocyte counts and Pax7+ and MyoD+ cells. Conclusions: Results suggest that extended football games induce a prolonged recovery of performance, which may be facilitated by carbohydrate supplementation during a congested game fixture. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of a far-infrared radiation (FIR) lamp therapy on changes in muscle damage and proprioception markers after maximal eccentric exercise of the elbow flexors (EF: Study 1) and the knee flexors (KF: Study 2) in comparison to a sham treatment condition. In each study, 24 healthy sedentary women were assigned to a FIR or a sham treatment group (n = 12/group). They performed 72 maximal EF eccentric contractions (Study 1) or 100 maximal KF eccentric contractions (Study 2) with their non-dominant limbs. They received a 30-min FIR (wavelength: 8–14 µm) or sham treatment at 1, 25, 49, 73 and 97 h post-exercise to the exercised muscles. Maximum voluntary isometric contraction (MVC) torque, muscle soreness, plasma creatine kinase activity, and proprioception assessed by position sense, joint reaction angle, and force match were measured before, and 0.5, 24, 48, 72, 96 and 120 h post-exercise. The outcome measures showed significant changes (P < 0.05) at 0.5-hour post-exercise (before treatment) similarly (P > 0.05) between the conditions in both studies. However, changes in all measures at 24–120 h post-exercise were smaller (P < 0.05) for the FIR than sham condition in both studies. For example, MVC torque returned to the baseline by 72 h post-exercise for the FIR condition in both studies, but was still 19 ± 6% (Study 1) or 17 ± 12% (Study 2) lower than the baseline at 120 h post-exercise for the sham condition. These results suggested that the FIR lamp therapy was effective for accelerating recovery from muscle damage. FIR lamp therapy was effective for recovering muscle strength and proprioception 1–3 days faster to the baseline. FIR lamp therapy was effective for attenuating muscle soreness by 55–60%, and reducing peak plasma CK activity by 45–89%. The effects of the FIR lamp therapy appear to be greater than other therapeutic interventions for eccentric exercise-induced muscle damage that have been investigated previously. [ABSTRACT FROM AUTHOR]

Background: Selective BRAF inhibitors, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been approved for treatment of metastatic melanomas with a BRAF p.V600E mutation. The clinical significance of non-codon 600 mutations remains unclear, in part, due to variation of kinase activity for different mutants.Methods: In this study, we categorized BRAF mutations according to the reported mutant kinase activity. A total of 1027 lung cancer, colorectal cancer or melanoma specimens were submitted for clinical mutation detection by next generation sequencing.Results: Non-codon 600 mutations were observed in 37% of BRAF-mutated tumors. Of all BRAF mutants, 75% were kinase-activated, 15% kinase-impaired and 10% kinase-unknown. The most common kinase-impaired mutant involves codon 594, specifically, p.D594G (c.1781A > G) and p.D594N (c.1780G > A). Lung cancers showed significantly higher incidences of kinase-impaired or kinase-unknown mutants. Kinase-impaired BRAF mutants showed a significant association with concomitant activating KRAS or NRAS mutations, but not PIK3CA mutations, supporting the reported interaction of these mutations.Conclusions: BRAF mutants with impaired or unknown kinase activity as well as concomitant kinase-impaired BRAF mutations and RAS mutations were detected in lung cancers, colorectal cancers and melanomas. Different therapeutic strategies based on the BRAF mutant kinase activity and the concomitant mutations may be worthwhile. [ABSTRACT FROM AUTHOR]

Despite remarkable initial responses of anaplastic lymphoma kinase (ALK) inhibitors in ALK-positive non-small cell lung cancer (NSCLC) patients, cancers eventually develop resistance within one to two years. This study aimed to compare the properties of iruplinalkib (WX‑0593) with other ALK inhibitors and report the comprehensive characterization of iruplinalkib against the crizotinib resistance. The inhibitory effect of iruplinalkib on kinase activity was detected. A kinase screen was performed to evaluate the selectivity of iruplinalkib. The effect of iruplinalkib on related signal transduction pathways of ALK and c-ros oncogene 1 (ROS1) kinases was examined. The cellular and in vivo activities of ALK inhibitors were compared in engineered cancer-derived cell lines and in mice xenograft models, respectively. Human hepatocytes derived from three donors were used for evaluating hepatic enzyme inducing activity. HEK293 cell lines expressing transportors were used to invesigated the drug interaction potential mediated by several transporters. The results showed iruplinalkib potently inhibited the tyrosine autophosphorylation of wild-type ALK, ALKL1196M, ALKC1156Y and epidermal growth factor receptor (EGFR)L858R/T790M. The inhibitory effects of iruplinalkib in patient-derived xenograft and cell line-derived xenograft models were observed. Moreover, iruplinalkib showed robust antitumor effects in BALB/c nude mice xenograft models with ALK-/ROS1-positive tumors implanted subcutaneously, and the tumor suppressive effects in crizotinib-resistant model was significantly better than that of brigatinib. Iruplinalkib did not induce CYP1A2, CYP2B6 and CYP3A4 at therapeutic concentration, and was also a strong inhibitor of MATE1 and MATE2K transporters, as well as P-gp and BCRP. In conclusion, iruplinalkib, a highly active and selective ALK/ROS1 inhibitor, exhibited strong antitumor effects in vitro and in crizotinib-resistant models. [ABSTRACT FROM AUTHOR]

Study Question: Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo?Summary Answer: Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium.What Is Known Already: Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls.Study Design, Size, Duration: In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 10 for both phases).Participants/materials, Setting, Methods: The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs.Main Results and the Role Of Chance: During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P < 0.05) and are further augmented following the 9-day treatment (P < 0.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P < 0.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation < 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P < 0.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P < 0.001) as well as in vitro decidualized ESCs (P < 0.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P < 0.05).Large Scale Data: Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243.Limitations, Reasons For Caution: The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples.Wider Implications Of the Findings: The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies-including the context of decidualization-related subfertility and infertility.Study Funding/competing Interest(s): This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article. [ABSTRACT FROM AUTHOR]

Simple Summary: Prostate cancer is a serious threat to male health worldwide; however, chemotherapy remains an urgent problem because of resistance and insensitivity to androgen-deprivation therapy. Therefore, discovering novel molecules and chemotherapy can provide a new strategy. Pyruvate kinase M2 (PKM2) is not only a vital enzyme in regulating cancer glycolysis, but is also a transcription that regulates gene expression through nuclear translocation, so the targeting PKM2 can be a promising therapy for prostate cancer. In this experiment, we found a novel specific PKM2 inhibitor—compound 3h—and conducted molecular and cellular experiments to evaluate its anticancer effect. Our results indicated that proliferation of LNCaP cells can be inhibited by compound 3h through inducing apoptosis and autophagy, and inhibiting glycolysis and mitochondria respiration. Therefore, our results provide a new way of developing novel chemotherapy for prostate cancer. Pyruvate kinase M2 (PKM2) is a key enzyme involved in the regulation of glycolysis. Although PKM2 is overexpressed in various tumor tissues, its functional role in cancer chemotherapy remains unexplored. In this study, we investigated the anticancer activity of a new PKM2 inhibitor, compound 3h, through the cell metabolism and associated signaling pathways in prostate cancer cells. To evaluate the molecular basis of specific PKM2 inhibitors, the interactions of compounds 3h and 3K with the PKM2 protein were assessed via molecular docking. We found that, compared to compound 3K, compound 3h exhibited a higher binding affinity for PKM2. Moreover, compound 3h significantly inhibited the pyruvate kinase activity and PKM2 expression. Cytotoxicity and colony formation assays revealed the potent anticancer activity of compound 3h against LNCaP cells. Compound 3h significantly increased the apoptotic and autophagic cell death in LNCaP cells. In addition, compound 3h induced AMPK activation along with the inhibition of the mTOR/p70S6K pathway. Furthermore, compound 3h significantly inhibited glycolysis and mitochondrial respiration, as determined by analyzing the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) production. Our results revealed that compound 3h caused apoptotic and autophagic cell death in LNCaP cells by inhibiting cancer cell metabolism. Therefore, blocking glycolytic pathways using specific PKM2 inhibitors can target cancer cell metabolism in PKM2-overexpressed prostate cancer cells. [ABSTRACT FROM AUTHOR]

Simple Summary: p90RSK is a downstream effector protein of the MAPK pathway. In many human cancers, the MAPK pathway is constitutively activated due to oncogenic mutations of its components. Consequently, p90RSK is hyperactive and capable of hyper phosphorylating substrates involved in tumorigenesis. p90RSK belongs to the AGC kinase family, which phosphorylates substrates with an RXRXXS/T consensus motif. MDM2 protein presents this consensus in its sequence at serine 166. MDM2 is a ubiquitin ligase that negatively regulates the protein stability of p53. The aim of this study has been to verify whether p90RSK is capable of phosphorylating MDM2 in serine 166 and to investigate the role of p90RSK in regulating the p53 pathway, especially in thyroid tumors in which the MAPK pathway is constitutively active and in which the development of specific drugs aimed at inhibiting the kinase activity of p90RSK could represent a new way of inhibiting tumorigenesis. The expression level of the tumor suppressor p53 is controlled by the E3 ubiquitin ligase MDM2 with a regulatory feedback loop, which allows p53 to upregulate its inhibitor MDM2. In this manuscript we demonstrated that p90RSK binds and phosphorylates MDM2 on serine 166 both in vitro and in vivo by kinase assay, immunoblot, and co-immunoprecipitation assay; this phosphorylation increases the stability of MDM2 which in turn binds p53, ubiquitinating it and promoting its degradation by proteasome. A pharmacological inhibitor of p90RSK, BI-D1870, decreases MDM2 phosphorylation, and restores p53 function, which in turn transcriptionally increases the expression of cell cycle inhibitor p21 and of pro-apoptotic protein Bax and downregulates the anti-apoptotic protein Bcl-2, causing a block of cell proliferation, measured by a BrdU assay and growth curve, and promoting apoptosis, measured by a TUNEL assay. Finally, an immunohistochemistry evaluation of primary thyroid tumors, in which p90RSK is very active, confirms MDM2 stabilization mediated by p90RSK phosphorylation. [ABSTRACT FROM AUTHOR]

The present study compared the ipsilateral repeated bout effect (IL‐RBE) and contralateral repeated bout effect (CL‐RBE) of the elbow flexors (EF) and knee flexors (KF) for the same interval between bouts to shed light on their mechanisms. Fifty‐two healthy sedentary young (20–28 years) men were randomly assigned to the IL‐EF, IL‐KF, CL‐EF, and CL‐KF groups (n = 13/group). Thirty maximal eccentric contractions of the EF were performed in IL‐EF and CL‐EF, and 60 maximal eccentric contractions of the KF were performed in IL‐KF and CL‐KF, with a 2‐week interval between bouts. Changes in muscle damage markers such as maximal voluntary contraction (MVC) torque, muscle soreness, and plasma creatine kinase activity, and proprioception measures before to 5 days post‐exercise were compared between groups. Changes in all variables were greater (p < 0.05) after the first than second bout for all groups, and the changes were greater (p < 0.05) for the EF than KF. The changes in all variables after the second bout were greater (p < 0.05) for the CL than IL condition for both EF and KF. The magnitude of the average protective effect was similar between CL‐EF (33%) and CL‐KF (32%), but slightly greater (p < 0.05) for IL‐EF (67%) than IL‐KF (61%). These demonstrate that the magnitude of CL‐RBE relative to IL‐RBE was similar between the EF and KF (approximately 50%), regardless of the greater muscle damage for the EF than KF. It appears that the CL‐RBE is more associated with neural adaptations at cerebrum, cerebellum, interhemispheric inhibition, and coricospinal tract, but the IL‐RBE is induced by additional adaptations at muscles. [ABSTRACT FROM AUTHOR]

Background: Inhalational anesthetics are known to disrupt PDZ2 domain-mediated protein-protein interactions of the postsynaptic density (PSD)-95 protein. The aim of this study is to investigate the underlying mechanisms in response to early isoflurane exposure on synaptic PSD-95 PDZ2 domain disruption that altered spine densities and cognitive function. The authors hypothesized that activation of protein kinase-G by the components of nitric oxide (NO) signaling pathway constitutes a mechanism that prevents loss of early dendritic spines and synapse in neurons and cognitive impairment in mice in response to disruption of PDZ2 domain of the PSD-95 protein.Methods: Postnatal day 7 mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active PSD-95 wild-type PDZ2 peptide or soluble guanylyl cyclase activator YC-1 along with their respective controls. Primary neurons at 7 days in vitro were exposed to isoflurane or PSD-95 wild-type PDZ2 peptide for 4 h. Coimmunoprecipitation, spine density, synapses, cyclic guanosine monophosphate-dependent protein kinase activity, and novel object recognition memory were assessed.Results: Exposure of isoflurane or PSD-95 wild-type PDZ2 peptide relative to controls causes the following. First, there is a decrease in PSD-95 coimmunoprecipitate relative to N-methyl-d-aspartate receptor subunits NR2A and NR2B precipitate (mean ± SD [in percentage of control]: isoflurane, 54.73 ± 16.52, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 51.32 ± 12.93, P = 0.001). Second, there is a loss in spine density (mean ± SD [spine density per 10 µm]: control, 5.28 ± 0.56 vs. isoflurane, 2.23 ± 0.67, P < 0.0001; and PSD-95 mutant PDZ2 peptide, 4.74 ± 0.94 vs. PSD-95 wild-type PDZ2 peptide, 1.47 ± 0.87, P < 0.001) and a decrease in synaptic puncta (mean ± SD [in percentage of control]: isoflurane, 41.1 ± 14.38, P = 0.001; and PSD-95 wild-type PDZ2 peptide, 50.49 ± 14.31, P < 0.001). NO donor or cyclic guanosine monophosphate analog prevents the spines and synapse loss and decline in the cyclic guanosine monophosphate-dependent protein kinase activity, but this prevention was blocked by soluble guanylyl cyclase or protein kinase-G inhibitors in primary neurons. Third, there were deficits in object recognition at 5 weeks (mean ± SD [recognition index]: male, control, 64.08 ± 10.57 vs. isoflurane, 48.49 ± 13.41, P = 0.001, n = 60; and female, control, 67.13 ± 11.17 vs. isoflurane, 53.76 ± 6.64, P = 0.003, n = 58). Isoflurane-induced impairment in recognition memory was preventable by the introduction of YC-1.Conclusions: Activation of soluble guanylyl cyclase or protein kinase-G prevents isoflurane or PSD-95 wild-type PDZ2 peptide-induced loss of dendritic spines and synapse. Prevention of recognition memory with YC-1, a NO-independent activator of guanylyl cyclase, supports a role for the soluble guanylyl cyclase mediated protein kinase-G signaling in countering the effects of isoflurane-induced cognitive impairment.Editor’s Perspective: [ABSTRACT FROM AUTHOR]

Background: Diabetes is a serious chronic metabolic disorder. Trichosanthes kirilowii Maxim. (TK) is traditionally used for the treatment of diabetes in traditional Chinese medicine (TCM). However, the clinical application of TK on diabetic patients and the hypoglycemic efficacies of TK are still unclear. Methods: A retrospective cohort study was conducted to analyze the usage of Chinese herbs in patients with type 2 diabetes in Taiwan. Glucose tolerance test was performed to analyze the hypoglycemic effect of TK. Proteomic approach was performed to identify the protein constituents of TK. Insulin receptor (IR) kinase activity assay and glucose tolerance tests in diabetic mice were further used to elucidate the hypoglycemic mechanisms and efficacies of TK. Results: By a retrospective cohort study, we found that TK was the most frequently used Chinese medicinal herb in type 2 diabetic patients in Taiwan. Oral administration of aqueous extract of TK displayed hypoglycemic effects in a dose-dependent manner in mice. An abundant novel TK protein (TKP) was further identified by proteomic approach. TKP interacted with IR by docking analysis and activated the kinase activity of IR. In addition, TKP enhanced the clearance of glucose in diabetic mice in a dose-dependent manner. Conclusions: In conclusion, this study applied a bed-to-bench approach to elucidate the hypoglycemic efficacies and mechanisms of TK on clinical usage. In addition, we newly identified a hypoglycemic protein TKP from TK. Our findings might provide a reasonable explanation of TK on the treatment of diabetes in TCM. [ABSTRACT FROM AUTHOR]

SEE GANDHI AND PLUN-FAVREAU DOI101093/AWW320 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: It has been postulated that heterozygous mutations in recessive Parkinson's genes may increase the risk of developing the disease. In particular, the PTEN-induced putative kinase 1 (PINK1) p.G411S (c.1231G>A, rs45478900) mutation has been reported in families with dominant inheritance patterns of Parkinson's disease, suggesting that it might confer a sizeable disease risk when present on only one allele. We examined families with PINK1 p.G411S and conducted a genetic association study with 2560 patients with Parkinson's disease and 2145 control subjects. Heterozygous PINK1 p.G411S mutations markedly increased Parkinson's disease risk (odds ratio = 2.92, P = 0.032); significance remained when supplementing with results from previous studies on 4437 additional subjects (odds ratio = 2.89, P = 0.027). We analysed primary human skin fibroblasts and induced neurons from heterozygous PINK1 p.G411S carriers compared to PINK1 p.Q456X heterozygotes and PINK1 wild-type controls under endogenous conditions. While cells from PINK1 p.Q456X heterozygotes showed reduced levels of PINK1 protein and decreased initial kinase activity upon mitochondrial damage, stress-response was largely unaffected over time, as expected for a recessive loss-of-function mutation. By contrast, PINK1 p.G411S heterozygotes showed no decrease of PINK1 protein levels but a sustained, significant reduction in kinase activity. Molecular modelling and dynamics simulations as well as multiple functional assays revealed that the p.G411S mutation interferes with ubiquitin phosphorylation by wild-type PINK1 in a heterodimeric complex. This impairs the protective functions of the PINK1/parkin-mediated mitochondrial quality control. Based on genetic and clinical evaluation as well as functional and structural characterization, we established p.G411S as a rare genetic risk factor with a relatively large effect size conferred by a partial dominant-negative function phenotype. [ABSTRACT FROM AUTHOR]

Background: Genetic variability in LRRK2 has been unequivocally established as a major risk factor for familial and sporadic forms of PD in ethnically diverse populations.Objectives: To resolve the role of LRRK2 in the Indian population.Methods: We performed targeted resequencing of the LRRK2 locus in 288 cases and 298 controls and resolved the haplotypic structure of LRRK2 in a combined cohort of 800 cases and 402 controls in the Indian population. We assessed the frequency of novel missense variants in the white and East Asian population by leveraging exome sequencing and densely genotype data, respectively. We did computational modeling and biochemical approach to infer the potential role of novel variants impacting the LRRK2 protein function. Finally, we assessed the phosphorylation activity of identified novel coding variants in the LRRK2 gene.Results: We identified four novel missense variants with frequency ranging from 0.0008% to 0.002% specific for the Indian population, encompassing armadillo and kinase domains of the LRRK2 protein. A common genetic variability within LRRK2 may contribute to increased risk, but it was nonsignificant after correcting for multiple testing, because of small cohort size. The computational modeling showed destabilizing effect on the LRRK2 function. In comparison to the wild-type, the kinase domain variant showed 4-fold increase in the kinase activity.Conclusions: Our study, for the first time, identified novel missense variants for LRRK2, specific for the Indian population, and showed that a novel missense variant in the kinase domain modifies kinase activity in vitro. © 2018 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Background: Immune system dysfunction, including higher levels of peripheral monocytes and inflammatory cytokines, is an important feature of Parkinson's disease (PD) pathogenesis, although the mechanism underlying the process remains to be investigated. In the central nervous system, it is well-known that α-synuclein (α-syn), a key protein involved in PD, activates microglia potently, and it is also reported that α-syn exists in the peripheral system, especially in erythrocytes or red blood cells (RBC) at exceedingly high concentration. The current study focused on the possibility that RBC-derived α-syn mediates the sensitization of peripheral monocytes in PD patients.Methods: The hyperactivation of monocytes was assessed quantitatively by measuring mRNA levels of typical inflammatory cytokines (including IL-1β, IL-6 and TNF-α) and protein levels of secreted inflammatory cytokines (including pro-inflammatory cytokines: IL-1β, IL-6, TNF-α, IL-8, IFN-γ, IL-2, and IL-12p70 and anti-inflammatory cytokines: IL-4, IL-10, and IL-13). Western blot, nanoparticle tracking analysis and electron microscopy were used to characterize RBC-derived extracellular vesicles (RBC-EVs). Inhibitors of endocytosis and leucine-rich repeat kinase 2 (LRRK2), another key protein involved in PD, were used to investigate how these two factors mediated the process of monocyte sensitization by RBC-EVs.Results: Increased inflammatory sensitization of monocytes was observed in PD patients and PD model mice. We found that α-syn-containing RBC-EVs isolated from PD model mice or free form oligomeric α-syn induced the inflammatory sensitization of THP-1 cells, and demonstrated that endocytosis was a requirement for this pathophysiological pathway. Furthermore, the hyperactivation of THP-1 cells induced by RBC-EVs was associated with increased LRRK2 production and kinase activity. The phenomenon of inflammatory sensitization of human monocytes and increased LRRK2 were also observed by the treatment of RBC-EVs isolated from PD patients.Conclusions: Our data provided new insight into how hyperactivation of monocytes occurs in PD patients, and identified the central role played by α-syn-containing RBC-EVs in this process. [ABSTRACT FROM AUTHOR]

Purpose: Osteosarcoma (OS) is a primary malignant bone tumor, and the cure rate has stagnated in the past three decades. Butein, a plant polyphenol extracted from many herbs, has been proved to possess anti-tumor activity. However, the effect of butein on human OS and the underlying mechanisms remain to be elucidated. Materials and Methods: The OS cell line 143B was used. The effects of butein were evaluated through the cell proliferation assay, flow cytometry, florescence and transmission electron microscopy, and western blotting. All statistical analyses were performed using GraphPad Prism 7.0. Results: Butein was found to inhibit cell proliferation by causing G2/ M phase arrest in the 143B cells. In addition, butein suppressed the invasion of 143B cells upon IL‐6 treatment. Additionally, we found that butein inhibited the invasion of 143B cells stimulated with IL-6 via the p-STAT3-MMP9 signaling pathway. Remarkably, butein triggered extrinsic and intrinsic apoptosis and autophagy of 143B cells. The process of autophagy may have tumor-supporting effects. Furthermore, butein induced oxidative stress as evidenced by ROS generation, increase in malondialdehyde (MDA) level, and decrease in GSH/GSSH ratio and GPX4 expression. N-acetylcysteine can reverse the change of ROS. Further experiments indicated apoptosis and autophagy could be attenuated by the N-acetyl-L-cysteine and c-Jun N-terminal kinase (JNK) inhibitor SP600125. Additionally, butein inhibited the Akt/mammalian target of rapamycin (mTOR) signaling pathway, and suppressed the Akt kinase activity increased apoptosis and autophagy. Conclusion: Our results revealed butein induced apoptosis and autophagy by regulating oxidative stress, activating the JNK signaling pathway and blocking the Akt/mTOR signaling pathway in OS cells. Additionally, butein inhibited the invasion of 143B cells stimulated with IL-6 through the pSTAT3- MMP9 signaling pathway. In view of these results, butein may be a potential anti-tumor drug targeting osteosarcoma. [ABSTRACT FROM AUTHOR]

This study investigated whether reduced-duration downhill walking (DW) would confer a protective effect against muscle damage induced by a subsequent bout of longer duration DW performed 1 week or 4 weeks later. Healthy young adults were allocated to a control or one of the preconditioning exercise (PRE-1wk or PRE-4wk) groups (10 men and 4 women per group). PRE-1wk and PRE-4wk groups performed 20-min DW (-28% slope, 5 km/h, 10% body mass added to a backpack) 1 week and 4 weeks before 40-min DW, respectively, and the control group performed 40-min DW only. Maximal voluntary contraction (MVC) knee extension torque, plasma creatine kinase (CK) activity, and muscle soreness (100-mm visual analog scale) were measured before, immediately after, and 24, 48, and 72 h after DW, and the changes in these variables were compared among groups. The control group showed symptoms of muscle damage (e.g., prolonged decrease in MVC: -14% ± 10% at 48 h post-DW) after 40-min DW. Changes in all variables after 40-min DW of PRE-1wk and PRE-4wk groups were 54%-61% smaller ( P < 0.05) than the control group, without significant differences between PRE-1wk and PRE-4wk groups for MVC and plasma CK activity. Importantly, changes after the preconditioning exercise (20-min DW) were 67%-69% smaller ( P < 0.05) than those after the 40-min DW of the control group. These findings suggest that 20-min DW resulting in minor muscle damage conferred a protective effect against subsequent 40-min DW, and its effect could last for more than 4 weeks. [ABSTRACT FROM AUTHOR]

Objectives: To examine whether an acute bout of active or dynamic hamstring-stretching exercises would reduce the amount of muscle damage observed after a strenuous eccentric task and to determine whether the stretching protocols elicit similar responses. Design: A randomized controlled clinical trial. Methods: Thirty-six young male students performed 5 min of jogging as a warm-up and were allocated to 1 of 3 groups: 3 min of static active stretching (SAS), 3 min of dynamic active stretching (DAS), or control (CON). All subjects performed eccentric exercise immediately after stretching. Heart rate, core temperature, maximal voluntary isometric contraction, passive hip flexion, passive hamstring stiffness (PHS), plasma creatine kinase activity, and myoglobin were recorded at prestretching, at poststretching, and every day after the eccentric exercises for 5 d. Results: After stretching, the change in hip flexion was significantly higher in the SAS (5°) and DAS (10.8°) groups than in the CON (–4.1°) group. The change in PHS was significantly higher in the DAS (5.6%) group than in the CON (–5.7%) and SAS (–6.7%) groups. Furthermore, changes in muscle-damage markers were smaller in the SAS group than in the DAS and CON groups. Conclusions: Prior active stretching could be useful for attenuating the symptoms of muscle damage after eccentric exercise. SAS is recommended over DAS as a stretching protocol in terms of strength, hamstring range of motion, and damage markers. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to compare pulmonary and plasma markers of oxidative stress and inflammation after concentric and eccentric cycling bouts in individuals with chronic obstructive pulmonary disease (COPD).Methods: Ten patients with moderate COPD level (68.3 ± 9.1 years; forced expiratory volume in 1 s = 68.6 ± 20.4% of predicted) performed 30 min of moderate-intensity concentric (CONC-M: 50% maximum concentric cycling power output; POmax) and eccentric cycling (ECC-M: 50% POmax), and high-intensity eccentric cycling (ECC-H: 100% POmax) in a randomised order. Cardiometabolic demand was monitored during cycling. Indirect markers of muscle damage were assessed before, immediately after, 24 and 48 h after cycling (muscle strength, muscle soreness and creatine kinase activity). Plasma oxidative stress (malondialdehyde: MDA), antioxidant (glutathione peroxidase activity: GPx) and inflammatory markers (IL-6, TNF-α) were measured before and 5 min after cycling. Exhaled breath condensate (EBC) samples were collected before and 15 min after cycling and analysed for hydrogen peroxide (H2O2), nitrites (NO2-) and pH.Results: Cardiometabolic demand was 40-50% lesser for ECC-M than CONC-M and ECC-H. Greater muscle damage was induced after ECC-H than ECC-M and CONC-M. MDA decreased immediately after CONC-M (- 28%), ECC-M (- 14%), and ECC-H (- 17%), while GPx remained unchanged. IL-6 increased only after ECC-H (28%), while TNF-α remained unchanged after exercise. Pulmonary H2O2, NO2- and pH remained unchanged after exercise.Conclusion: These results suggest that only moderate muscle damage and inflammation were induced after high-intensity eccentric cycling, which did not induce pulmonary or plasmatic increases in markers of oxidative stress.Trial Registration Number: Trial registration number: DRKS00009755. [ABSTRACT FROM AUTHOR]

Epidermal growth factor (EGF) has been found to stimulate proliferation in a variety of cell types. The EGF receptor is known to have tyrosine kinase activity [1], however, the role of this signal mechanism has not been established in bone cells. The aim of this study was to determine whether tyrosine kinase activity and G inhibitory (Gi) proteins are involved in EGF-stimulated proliferation in the osteoblastic cell line G292 and in primary culture osteoblasts isolated from neonatal rat calvaria. Cell proliferation was measured by 3H-thymidine incorporation using liquid scintillation spectrometry. EGF stimulates a dose-dependent increase in proliferation of G292 and primary culture cells above control. Genistein was able to inhibit the effects of EGF in the G292 cells. In the primary culture cells, genistein with EGF appeared to enhance proliferation compared with EGF alone or genistein alone. Tyrphostin 25, on the other hand, inhibited the EGF response in both of these cell types. Inactivation of Gi proteins with pertussis toxin was able to inhibit EGF-induced mitogenesis in the neonatal rat osteoblasts but did not appear to specifically inhibit this response in the G292 cells. These results suggest that although both of these osteoblastic cell types increase proliferation in response to EGF, their signal pathways are different. [ABSTRACT FROM AUTHOR]

Introduction: The present study tested the hypothesis that eccentric training (ET) of nonimmobilized arm would attenuate negative effects of immobilization and provide greater protective effects against muscle damage induced by eccentric exercise after immobilization, when compared with concentric training (CT). Methods: Sedentary young men were placed to ET, CT, or control group (n = 12 per group), and their nondominant arms were immobilized for 3 wk. During the immobilization period, the ET and CT groups performed five sets of six dumbbell curl eccentric-only and concentric-only contractions, respectively, at 20%–80% of maximal voluntary isometric contraction (MVCiso) strength over six sessions. MVCiso torque, root-mean square (RMS) of electromyographic activity during MVCiso, and bicep brachii muscle cross-sectional area (CSA) were measured before and after immobilization for both arms. All participants performed 30 eccentric contractions of the elbow flexors (30EC) by the immobilized arm after the cast was removed. Several indirect muscle damage markers were measured before, immediately after, and for 5 d after 30EC. Results: ET increased MVCiso (17% ± 7%), RMS (24% ± 8%), and CSA (9% ± 2%) greater (P < 0.05) than CT (6% ± 4%, 9% ± 4%, 3% ± 2%) for the trained arm. The control group showed decreases in MVCiso (−17% ± 2%), RMS (−26% ± 6%), and CSA (−12% ± 3%) for the immobilized arm, but these changes were attenuated greater (P < 0.05) by ET (3% ± 3%, −0.1% ± 2%, 0.1% ± 0.3%) than CT (−4% ± 2%, −4% ± 2%, −1.3% ± 0.4%). Changes in all muscle damage markers after 30EC were smaller (P < 0.05) for the ET and CT than the control group, and ET than the CT group (e.g., peak plasma creatine kinase activity: ET, 860 ± 688 IU·L−1; CT, 2390 ± 1104 IU·L−1; control, 7819 ± 4011 IU·L−1). Conclusions: These results showed that ET of the nonimmobilized arm was effective for eliminating the negative effects of immobilization and attenuating eccentric exercise–induced muscle damage after immobilization. [ABSTRACT FROM AUTHOR]

Background. Rho guanine nucleotide exchange factor 10-like protein (ARHGEF10L) is a member of the guanine nucleotide exchange factor family, which regulates Rho GTPase activities, thus contributing to tumorigenesis. Our previous study demonstrated a strong association between the ARHGEF10L gene and the risk of cervical carcinoma. This study investigated the pathogenic role and mechanism of ARHGEF10L in cervical tumors. Methods. The HeLa cell line, which was derived from cervical carcinoma, was transfected with ARHGEF10L-overexpressing plasmids or anti-ARHGEF10L siRNA. Cell counting kit-8 assays, wound-healing assays, and cell apoptosis assays were performed to investigate the effects of ARHGEF10L on cell activities. A Rho pull-down assay and RNA-sequencing analysis were performed to investigate the pathogenic pathway of ARHGEF10L involvement in cervical tumors. Results. ARHGEF10L overexpression promoted cell proliferation and migration, reduced cell apoptosis, and induced epithelial-to-mesenchymal transition (EMT) via downregulation of E-cadherin and upregulation of N-cadherin and Slug in transfected HeLa cells. The overexpression of ARHGEF10L also upregulated GTP-RhoA, ROCK1, and phospho-ezrin/radixin/moesin (ERM) expression in HeLa cells. RNA-sequencing analysis detected altered transcription of 31 genes in HeLa cells with ARHGEF10L overexpression. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway analyses identified significant differences in cyclin-dependent protein serine/threonine kinase activity, cell responses to vitamin A, and Toll-like receptor signaling pathways. Both real-time PCR and Western blotting verified the increased expression of heat shock 70 kDa protein 6 (HSPA6) in ARHGEF10L-overexpressing HeLa cells. Since we reported that ARHGEF10L played a role through RhoA-ROCK1-ERM signaling, an important pathway in tumorigenesis, and stimulated EMT and HSPA6 expression in liver tumors and gastric tumor cells, we suggest that ARHGEF10L is a novel oncogene in many tumors. [ABSTRACT FROM AUTHOR]