Concomitant Signaling Research Articles

Syndesmotic and Deltoid Injuries: Companions or Coincidences.

Syndesmotic complex injuries are supposedly associated with injuries to the deltoid ligament (DL) complex. Several syndesmosis classifications take DL injuries into account when rating the stability of the syndesmotic injury. Still, no study has yet assessed the frequency and severity of DL injuries in unstable syndesmotic injuries. The aim of this retrospective cohort study was to assess both the severity of the syndesmotic and DL injury in patients undergoing surgery for an unstable syndesmotic injury. The integrity of the syndesmotic and DL complex of 37 patients were assessed on MRI by 2 musculoskeletal radiologists. Eligible were adult patients with an acute, isolated, unstable syndesmotic injury (ligamentous or bony avulsions) who underwent surgery. The DL complex was classified as having 3 superficial (TNL/TSL/TCL) and 2 deep (aTTL/pTTL) components. Grade 0 indicated an intact ligament; grade I showed a periligamentous edema; grade II a partial tear presenting as laxity, irregular contour, or partial discontinuity with concomitant hyperintense signaling; grade III a complete tear; or grade IV in case of an avulsion fracture. Frequency and severity of DL injuries were assessed, and possible injury combinations analyzed using an h-cluster analysis and Mann-Whitney U test. Mean severity (grade 0-3) was 2.6 ± 0.8 for the syndesmotic complex and 1.6 ± 1.1 for the DL (superficial 1.5 ± 1.1, deep 1.6 ± 1.1). Seven patients (19%) had no (n = 3; 8%) or minor (grade 1: n = 4; 11%) DL injuries. Overall, 2 different patient clusters and 4 separate ligament clusters were identified. Unstable syndesmotic injuries can occur isolated or with an accompanying DL injury. There appears to be a great heterogeneity between the severity of syndesmotic and deltoid ligament injuries.

The anti-neoplastic impact of thymoquinone from Nigella sativa on small cell lung cancer: In vitro and in vivo investigations.

Malignant and aggressive, small cell lung cancer (SCLC) constitutes about 15% of all diagnosed lung cancer cases. With primary therapeutic options such as chemotherapy accompanied by debilitating side effects, interest has been soaring in the therapeutic competencies of herbs. The pharmacological driving force behind the beneficial properties of Nigella sativa is the quinone, thymoquinone (TQ). The anti-cancer effects of TQ on different cancers have been extensively studied. Nonetheless, only one paper in the entire National Center for Biotechnology Information (NCBI) database describes its effects on SCLC. A more detailed investigation is required. The current study examined the impact of TQ in vitro on five SCLC cell lines and in vivo in a nude mouse xenograft model. The following in vitro effects of TQ on SCLC were evaluated: (a) cell viability; (b) apoptosis; (c) cell cycle arrest; (d) intracellular reactive oxygen species (ROS) levels, and (e) protein expression in concomitant signaling pathways. For the in vivo effects of TQ on SCLC, (a) tumor volume was measured, and (b) selected protein expression in selected concomitant signaling pathways was determined by Western blotting. In general, TQ reduced cell viability, induced apoptosis and cell cycle arrest, depleted ROS, and altered protein expression in associated signaling pathways. Furthermore, TQ exhibited a tumor-suppressive effect in an H446 SCLC xenograft model. The cytotoxic impact of TQ arising from anti-cancer mechanisms was elucidated. The positive results obtained in this study warrant further investigation.

Mild Osmotic Stress Alters mTOR Pathway Signaling

Increased osmotic stress reduces cell volume, thus disrupting homeostatic processes. This could impact signaling that promotes muscle growth and maintenance at rest and in response to mechanical stimuli. To date, the detrimental effect of osmotic stress on muscle protein turnover has been established in in vitro and in situ models, but the effects on muscular anabolic signaling in humans are unknown. Purpose: To examine the effect of fluid restriction (FR)-induced mild osmotic stress (plasma osmolality ≥295.0 mmol×kg−1) on muscular anabolic signaling in response to resistance exercise (RE) in trained young men. Hypothesis: We hypothesized that FR would alter the Akt/mTOR signaling. Method: RE-trained men (n=11, 21±1yr, 175.9±6.2cm, 79.2±12.3kg, 18.4±6.7% fat) completed 2 identical RE bouts either with (FR) or without (CON) fluid restriction from 24 hrs before to 3 hrs after RE. RE consisted of bilateral leg press and knee extensions (5 sets x 10 repetitions at 80% of 1 repetition maximum). The two conditions were 2-3 wks apart. Plasma osmolality (PO) was measured before (PRE), and 5 min, 15 min, 30 min, 1 hr, and 3 hrs after RE. Muscle samples were collected at PRE and 1 and 3 hrs after RE. Western blots were used to analyze contents of proteins in the mTOR signaling pathway: Akt, p-AktS473, mTOR, p-mTORS2448, p70S6K, p-p70S6KT389, rpS6, p-rpS6S240/244, eEF2, and p-eEF2T56 and normalized to Ponceau S staining (total protein). Results: Increased PO was successfully induced in FR vs. CON (condition effect, p<0.0001; 299±2 vs 286±1 mmol/kg). P-AktS473 significantly (p≤0.05) increased from PRE to 1 and 3 hrs in CON, and from PRE to 1h with a return to PRE by 3h in FR (condition x time). P-AktS473 was higher at 1h and lower at 3h in FR versus CON. P-mTORS2448 increased from PRE to 1 and 3 hrs regardless of condition (main time). P-p70S6KT389 was greater in FR than CON regardless of time point (main condition). P-rpS6S240/244 increased from PRE to 1 and 3 hrs for CON and FR, and was greater for FR than CON at PRE (condition x time). Conclusion: Baseline anabolic signaling appeared elevated when individuals were subjected to mild osmotic stress. After RE, signaling through mTOR was elevated as expected when plasma was iso-osmotic, although anticipated time effects for p-p70S6K and p-eEF2 were not detected. Upstream of mTOR, activation of Akt was stronger yet shorter in duration with mild osmotic stress. Downstream of mTOR, p70S6K and rpS6 activation were greater with mild osmotic stress. While these results suggest increased anabolic signaling with mild osmotic stress, concomitant catabolic signaling should be assessed. Together, the present results and future measures will inform how conditions that confer osmotic stress, such as dehydration, may alter intramuscular protein homeostasis at rest and in response to an anabolic stimulus. This study was funded by National Strength and Conditioning Association Foundations. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pre-T cell receptor localization and trafficking are independent of its signaling.

Expression of the pre-T cell receptor (preTCR) is an important checkpoint during the development of T cells, an essential cell type of our adaptive immune system. The preTCR complex is only transiently expressed and rapidly internalized in developing T cells and is thought to signal in a ligand-independent manner. However, identifying a mechanistic basis for these unique features of the preTCR compared with the final TCR complex has been confounded by the concomitant signaling that is normally present. Thus, we have reconstituted preTCR expression in non-immune cells to uncouple receptor trafficking dynamics from its associated signaling. We find that all the defining features of the preTCR are intrinsic properties of the receptor itself, driven by exposure of an extracellular hydrophobic region, and are not the consequence of receptor activation. Finally, we show that transitory preTCR cell surface expression can sustain tonic signaling in the absence of ligand binding, suggesting how the preTCR can nonetheless drive αβTCR lineage commitment.

3-Deoxysappanchalcone Inhibits Cell Growth of Gefitinib-Resistant Lung Cancer Cells by Simultaneous Targeting of EGFR and MET Kinases.

The mechanistic functions of 3-deoxysappanchalcone (3-DSC), a chalcone compound known to have many pharmacological effects on lung cancer, have not yet been elucidated. In this study, we identified the comprehensive anti-cancer mechanism of 3-DSC, which targets EGFR and MET kinase in drug-resistant lung cancer cells. 3-DSC directly targets both EGFR and MET, thereby inhibiting the growth of drug-resistant lung cancer cells. Mechanistically, 3-DSC induced cell cycle arrest by modulating cell cycle regulatory proteins, including cyclin B1, cdc2, and p27. In addition, concomitant EGFR downstream signaling proteins such as MET, AKT, and ERK were affected by 3-DSC and contributed to the inhibition of cancer cell growth. Furthermore, our results show that 3-DSC increased redox homeostasis disruption, ER stress, mitochondrial depolarization, and caspase activation in gefitinib-resistant lung cancer cells, thereby abrogating cancer cell growth. 3-DSC induced apoptotic cell death which is regulated by Mcl-1, Bax, Apaf-1, and PARP in gefitinib-resistant lung cancer cells. 3-DSC also initiated the activation of caspases, and the pan-caspase inhibitor, Z-VAD-FMK, abrogated 3-DSC induced-apoptosis in lung cancer cells. These data imply that 3-DSC mainly increased mitochondria-associated intrinsic apoptosis in lung cancer cells to reduce lung cancer cell growth. Overall, 3-DSC inhibited the growth of drug-resistant lung cancer cells by simultaneously targeting EGFR and MET, which exerted anti-cancer effects through cell cycle arrest, mitochondrial homeostasis collapse, and increased ROS generation, eventually triggering anticancer mechanisms. 3-DSC could potentially be used as an effective anti-cancer strategy to overcome EGFR and MET target drug-resistant lung cancer.

PSGL-1 deficiency re-wires T cell signaling to support development of stem cell like exhausted T cells that control PD-1 blockade resistant melanoma tumor growth

Abstract We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates CD8 +T cell effector functions and promotes exhaustion in responses to chronic viral infection and melanoma tumors. In this study, we investigated cellular and molecular mechanisms by which intrinsic PSGL-1 signaling regulates the differentiation of exhausted T cells (T EX). We show that PSGL-1 localizes with the TCR upon co-ligation and find that PSGL-1 associates with Zap70 and the Zap70 inhibitor, Sts-1. In the absence of PSGL-1, expression of Sts-1 is reduced allowing for greater activation of Zap70 and the downstream signaling molecules Akt and Erk1/2. In addition, PSGL-1-deficiency empowers CD8 +T cells to respond to low affinity TCR ligands. In vivo, PSGL-1 deficiency leads to growth inhibition of a PD-1-blockade resistant melanoma. Notably, PSGL-1-deficiency in T cells enables retention of effector T cell (T EFF) functions, and an increased representation of stem cell-like T cells (T SC) that express TCF-1 and low levels of TOX, which are associated with sustained effector responses and responsiveness to ICB. We now show that concomitant PSGL-1 signaling with repeated TCR stimulation of human T EFFelicits functional exhaustion. Importantly, blockade of PSGL-1 recapitulates PSGL-1 deficiency in anti-tumor as well as antiviral responses, and its distinct mechanism of T cell inhibition underscores the translational potential of targeting PSGL-1 by ICB. Supported by grants from NIH (R01 AI106895, R21 CA249353, R21 CA216678, R03 CA252144) Melanoma Research Alliance MRA 696326, Department of Defense W81XWH-20-1-0324, Sanford Burnham Prebys NCI-Designated Cancer Center Support Grant, P30 CA030199 American Cancer Society Postdoctoral Fellowship (PF-20-113-01-LIB)

PI(4,5)P2-dependent and -independent roles of PI4P in the control of hormone secretion by pituitary cells.

Plasma membrane and organelle membranes are home to seven phosphoinositides, an important class of low-abundance anionic signaling lipids that contribute to cellular functions by recruiting cytoplasmic proteins or interacting with the cytoplasmic domains of membrane proteins. Here, we briefly review the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cellular signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, acting as a substrate for phospholipase C, plays a key role in the control of pituitary cell functions, including hormone synthesis and secretion. PI(4,5)P2 also acts as a substrate for class I PI3-kinases, leading to the generation of two intracellular messengers, PI(3,4,5)P3 and PI(3,4)P2, which act through their intracellular effectors, including Akt. PI(4,5)P2 can also influence the release of pituitary hormones acting as an intact lipid to regulate ion channel gating and concomitant calcium signaling, as well as the exocytic pathway. Recent findings also show that PI4P is not only a precursor of PI(4,5)P2, but also a key signaling molecule in many cell types, including pituitary cells, where it controls hormone secretion in a PI(4,5)P2-independent manner.

Longterm Increased S100B Enhances Hippocampal Progenitor Cell Proliferation in a Transgenic Mouse Model.

(1) The neurotrophic protein S100B is a marker of brain injury and has been associated with neuroregeneration. In S100Btg mice rendering 12 copies of the murine S100B gene we evaluated whether S100B may serve as a treatment option. (2) In juvenile, adult, and one-year-old S100Btg mice (female and male; n = 8 per group), progenitor cell proliferation was quantified in the subgranular zone (SGZ) and the granular cell layer (GCL) of the dentate gyrus with the proliferative marker Ki67 and BrdU (50 mg/kg). Concomitant signaling was quantified utilizing glial fibrillary acidic protein (GFAP), apolipoprotein E (ApoE), brain-derived neurotrophic factor (BDNF), and the receptor for advanced glycation end products (RAGE) immunohistochemistry. (3) Progenitor cell proliferation in the SGZ and migration to the GCL was enhanced. Hippocampal GFAP was reduced in one-year-old S100Btg mice. ApoE in the hippocampus and frontal cortex of male and BDNF in the frontal cortex of female S100Btg mice was reduced. RAGE was not affected. (4) Enhanced hippocampal neurogenesis in S100Btg mice was not accompanied by reactive astrogliosis. Sex- and brain region-specific variations of ApoE and BDNF require further elucidations. Our data reinforce the importance of this S100Btg model in evaluating the role of S100B in neuroregenerative medicine.

Exploration of new therapeutic targets for viral hepatic fibrosis, alcoholic hepatic fibrosis, and non-alcoholic hepatic fibrosis.

BackgroundHepatic fibrosis is a widespread disease worldwide. Millions of people lose their lives due to hepatic fibrosis every year. The main causes of hepatic fibrosis include viral infection, alcoholism, and obesity. Many studies have been conducted on the single factors that cause hepatic fibrosis; however, no studies have examined whether hepatic fibrosis caused by multiple factors has concomitant expression molecules and signaling pathways. In this study, we sought to analyze the common differentially expressed messenger ribonucleic acids (mRNAs) of hepatic fibrosis caused by different factors, including hepatitis B virus (HBV) hepatic fibrosis, alcoholic hepatic fibrosis, and non-alcoholic hepatic fibrosis, and identify potential preventive and therapeutic targets.MethodsThe GSE171294, GSE142530, and GSE126848 datasets from the Gene Expression Omnibus (GEO) public database were used in this study. A |log fold change| >0.5 and a P value <0.05 were defined as differentially expressed mRNAs via R software screening. To further screen the target mRNAs, the differential mRNAs were subjected to a functional enrichment analysis based on the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Finally, the relationships between differentially expressed mRNA-encoded proteins were analyzed by a protein-protein interaction (PPI) analysis.ResultsA total of 54 differentially expressed mRNAs were identified. The KEGG analysis showed that the functions of different mRNAs mainly focused on Gonadotropin Releasing Hormone (GnRH) secretion, bile secretion and insulin secretion. The GO enrichment analysis showed that the differential mRNAs were mainly present in the cytoplasmic membrane region and exerted biological functions, such as activating channels and binding proteins by regulating biological processes (BPs), such as cells, cytoskeleton and heparin. The PPI network analysis revealed 16 nodes with 12 pairs of interactions. The 16 critical nodes included BCL6, CD4, CD24, IL32, CALD1, TRAF3, SOX9, KANSL3, MRGBP, PKD2, PKHD1, SYT1, ANXA4, KCNMA1, KCNN2, and CACNA1H.ConclusionsKCNN2, CD4, CD24, BCL6, KCNMA1, and other molecules obtained by the bioinformatics analysis of the RNA-sequencing data can be used as new research targets for hepatic fibrosis induced by different causes. Our findings could provide novel ideas for the treatment of hepatic fibrosis.

The Role of Tumor Necrosis Factor Associated Factors (TRAFs) in Vascular Inflammation and Atherosclerosis.

TNF receptor associated factors (TRAFs) represent a family of cytoplasmic signaling adaptor proteins that regulate, bundle, and transduce inflammatory signals downstream of TNF- (TNF-Rs), interleukin (IL)-1-, Toll-like- (TLRs), and IL-17 receptors. TRAFs play a pivotal role in regulating cell survival and immune cell function and are fundamental regulators of acute and chronic inflammation. Lately, the inhibition of inflammation by anti-cytokine therapy has emerged as novel treatment strategy in patients with atherosclerosis. Likewise, growing evidence from preclinical experiments proposes TRAFs as potent modulators of inflammation in atherosclerosis and vascular inflammation. Yet, TRAFs show a highly complex interplay between different TRAF-family members with partially opposing and overlapping functions that are determined by the level of cellular expression, concomitant signaling events, and the context of the disease. Therefore, inhibition of specific TRAFs may be beneficial in one condition and harmful in others. Here, we carefully discuss the cellular expression and signaling events of TRAFs and evaluate their role in vascular inflammation and atherosclerosis. We also highlight metabolic effects of TRAFs and discuss the development of TRAF-based therapeutics in the future.

Identification of functional pathways for regenerative bioactivity of selected renal cells

BackgroundSelected renal cells (SRC) are in Phase II clinical trials as a kidney-sourced, autologous, tubular epithelial cell-enriched cell-based therapy for chronic kidney disease (CKD). In preclinical studies with rodent models of CKD, SRC have been shown to positively modulate key renal biomarkers associated with development of the chronic disease condition.MethodsA comparative bioinformatic analysis of transcripts specifically enriched or depleted in SRC component sub-populations relative to the initial, biopsy-derived cell source was conducted.ResultsOutcomes associated with therapeutically relevant bioactivity from a systematic, genome-wide transcriptomic profiling of rodent SRC are reported. Key transcriptomic networks and concomitant signaling pathways that may underlie SRC mechanism of action as manifested by reparative, restorative, and regenerative bioactivity in rodent models of chronic kidney disease are identified. These include genes and gene networks associated with cell cycle control, transcriptional control, inflammation, ECM–receptor interaction, immune response, actin polymerization, regeneration, cell adhesion, and morphogenesis.ConclusionsThese data indicate that gene networks associated with development of the kidney are also leveraged for SRC regenerative bioactivity, providing evidence of potential mechanisms of action.

Abstract 13123: Global Knockout of LDLRAP1 Regulates Atherosclerosis, Adipose Metabolism, and Insulin Resistance

Introduction: Atherosclerotic vascular syndromes account for 50% of all deaths in the United States, and are a considerable medical and socioeconomic problem contributing to mortality of several conditions, including myocardial infarction and stroke. It is accepted that atherosclerosis and symptoms of metabolic syndrome, such as obesity, insulin resistance, and hypercholesterolemia often coincide with and exacerbate one another, however the molecular events in common with these conditions are not yet fully understood. One event that may link these two disorders is lipid receptor trafficking and signaling of the LDL receptor (LDLR), which plays a key role in LDL uptake and regulation of plasma LDL concentration. Low-density lipoprotein receptor adaptor protein 1 (LDLRAP1) is a cytosolic adaptor protein which interacts with the cytoplasmic tail of the LDLR, mediating internalization of the LDL-LDLR complex and connecting it to the cell’s endocytic machinery. The purpose of this study was to characterize the effects of LDLRAP1 global knockout on atherogenesis and metabolic function in order to gain a better understanding of the molecular mechanisms that interconnect atherosclerosis and metabolic syndrome. Results: LDLRAP1 global knockout mice fed a high-fat western diet (HFD) for 16 weeks displayed increased atherosclerotic plaque burden (p&lt;0.0001), serum cholesterol (p&lt;0.0001) and triglyceride levels (p&lt;0.01), and expression of lipid receptors SR-A1 and CD36 (p&lt;0.05), and gained significantly more weight compared to controls (p&lt;0.0001). LDLRAP1 -/- adipose also displayed decreased glucose uptake (p&lt;0.01) and pAKT and AKT protein levels (p&lt;0.05). Both before and after a HFD, LDLRAP1 -/- mice were also significantly insulin resistant (p&lt;0.01) with concomitant dysregulated insulin signaling in adipose tissue, and displayed metabolic differences as quantified by metabolic cage analysis (p&lt;0.05). Conclusions: These data suggest that in addition to a function in cholesterol internalization, LDLRAP1 participates in regulating adipose metabolism and insulin sensitivity. This implicates LDLRAP1 as a key molecule and potential therapeutic target in treatment of diseases which constitute metabolic syndrome.

Characterization of functional protein complexes from Alzheimer\u2019s disease and healthy brain by mass spectrometry-based proteome analysis

Alzheimer’s disease (AD) is a complex neurodegenerative disorder with impaired protein activities. Proteins in the form of complexes have a ubiquitous role in diverse range of cellular functions. The key challenge is to identify novel disease associated protein complexes and their potential role in the progression of AD pathology. Protein complexes were obtained from AD brain prefrontal cortex and age matched controls by Blue Native-Polyacrylamide Gel Electrophoresis. A proteomic analysis was performed using second dimension SDS-PAGE followed by nano LC–MS/MS. Differentially expressed proteins were mapped to existing biological networks by Ingenuity Pathway Analysis (IPA). A total of 13 protein complexes with their interacting proteins were resolved on SDS-PAGE. We identified 34 protein spots and found significant abundance difference between the two experimental samples. IPA analysis revealed degeneration of neurons and cell death as a major consequence of protein dysregulation. Furthermore, focused network analysis suggested an integrated regulation of the identified proteins through APP and MAPT dependent mechanisms. The interacting differentially expressed proteins in AD were found to be part of concomitant signaling cascades terminating in neuronal cell death. The identified protein networks and pathways warrant further research to study their actual contribution to AD pathology.

Porphyromonas gingivalis promotes tumor progression in esophageal squamous cell carcinoma.

Increasing evidence indicates that the microbiome may influence tumor growth and modulate the tumor microenvironment of gastrointestinal cancers. However, the role of oral bacteria in the development of esophageal squamous cell carcinoma (EsoSCC) has remained unclear. Herein, we investigated the relationship between the periodontal pathogen Porphyromonas gingivalis and EsoSCC. To identify bacterial biomarkers associated with EsoSCC, we analyzed microbiomes in oral biofilms. The presence of P. gingivalis in esophageal tissues and relationships of P. gingivalis infection with clinicopathologic characteristics in 156 patients with EsoSCC were assessed using immunohistochemistry. The role of P. gingivalis infection in in vitro and in vivo EsoSCC progression was also assessed. Microbiota profiles in oral biofilms revealed that P. gingivalis abundance was associated with an increased risk of EsoSCC development. In total, 57% of patients with EsoSCC were found to be infected with P. gingivalis. The presence of P. gingivalis was found to be associated with advanced clinical stages and a poor prognosis. It was also found to be associated with an elevated esophageal cancer incidence in a 4-nitroquinoline 1-oxide-induced mouse model andwith an increased xenograft tumor growth. P. gingivalis infection increased interleukin (IL)-6 production and it promoted epithelial-mesenchymal transition and the recruitment of myeloid-derived suppressor cells. Furthermore, inhibited IL-6 signaling attenuated the tumor-promoting effects of P. gingivalis in 4-nitroquinoline 1-oxide-treated mice and xenograft mouse models. Our data indicate that P. gingivalis may promote esophageal cancer development and progression. Direct targeting of P. gingivalis or concomitant IL-6 signaling may be a promising strategy to prevent and/or treat EsoSCC associated with P. gingivalis infection.

Concomitant aerobic- and hypertrophy-related skeletal muscle cell signaling following blood flow-restricted walking

This study is aimed to examine the acute responses to a blood flow restriction (BFR) low-intensity aerobic exercise, compared with a similar exercise without BFR, on several aerobic- and hypertrophy-related proteins expression and hormonal responses. On two different occasions, five healthy untrained male subjects were required to perform (i) a BFR low-intensity aerobic exercise consisting of 5 sets of 2-min bouts of walking at an exercise intensity of 63–65% HRmax (equivalent to 40% of VO2max), interspersed by 1-min rest; and (ii) similar exercise bouts without BFR (Ctrl). For each condition, venous blood samples were collected at pre- (baseline), immediately and 2-h post-exercise. Baseline and 3-h post-exercise muscle biopsy samples (vastus lateralis) were also performed for protein expression analysis. HIF-1α, PGC-1α, and VEGF proteins content were significantly higher (P < 0.05) at 3-h post-exercise in BFR compared with baseline values. In reference to baseline, phosphorylation of Akt also increased significantly (P < 0.05) following BFR, and in a greater extent (P < 0.05) than following Ctrl. IGF-1 concentration significantly increased (P = 0.001) immediately following BFR exercise than baseline values and serum GH showed a significant increase (P = 0.046) compared with Ctrl. The addition of blood flow restriction during walking exercise initiate a concomitant cell signaling pathways regulating mitochondrial biogenesis, angiogenesis, and skeletal muscle protein expression. Le but de cette étude est d’examiner les réponses aiguës à un exercice aérobie de faible intensité réalisé avec ou sans occlusion vasculaire (blood flow restriction, [BFR]) sur l’expression de protéines impliquées dans le développement de l’hypertrophie et de l’endurance. Selon un plan croisé, 5 sujets sains non entraînés ont réalisé à deux occasions différentes: (i) 5 séries de 2 min de marche à 63–65 % de la fréquence cardiaque maximale (équivalent à 40 % de la consommation maximale d’oxygène) entrecoupées de 1 min de repos, le tout sous occlusion vasculaire des membres inférieurs (BFR); et (ii) le même exercice sans occlusion (Ctrl). Pour chaque condition, des échantillons sanguins ont été prélevés avant, immédiatement après et 2 h après chaque condition. Des biopsies musculaires (vastus lateralis) ont également été pratiquée au repos (avant exercice) et 3 h après. Le contenu protéique du facteur induit par l’hypoxie 1 alpha (Hypoxic Inducible Factor 1α, HIF-1α), de peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), et du facteur de croissance de l’endothélium vasculaire (Vascular Endothelial Growth Factor, VEGF) ont significativement augmenté (p < 0,05) après l’exercice aérobie BFR. La phosphorylation de la protéine kinase B (Akt) a également augmenté de manière significative (p < 0,05) après l’exercice aérobie BFR et dans une plus grande proportion (p < 0,05) que suite à l’exercice Ctrl. La concentration du facteur de croissance Insulin-like Growth Factor-1 (IGF-1) augmente (p < 0,001) immédiatement après l’exercice aérobie BFR et l’hormone de croissance (sérum GH) augmente significativement (p < 0,05) en comparaison avec Ctrl. L’ajout d’une restriction de la circulation sanguine au cours d’un exercice de marche stimule des voies de signalisation musculaire concomitantes régulant la biogenèse mitochondriale, l’angiogenèse et l’expression des protéines du muscle squelettique.

NK cells are activated and primed for skin-homing during acute dengue virus infection in humans

Despite animal models showing that natural killer (NK) cells are important players in the early defense against many viral infections, the NK cell response is poorly understood in humans. Here we analyze the phenotype, temporal dynamics, regulation and trafficking of NK cells in a patient cohort with acute dengue virus infection. NK cells are robustly activated and proliferate during the first week after symptom debut. Increased IL-18 levels in plasma and in induced skin blisters of DENV-infected patients, as well as concomitant signaling downstream of the IL-18R, suggests an IL-18-dependent mechanism in driving the proliferative NK cell response. Responding NK cells have a less mature phenotype and a distinct chemokine-receptor imprint indicative of skin-homing. A corresponding NK cell subset can be localized to skin early during acute infection. These data provide evidence of an IL-18-driven NK cell proliferation and priming for skin-homing during an acute viral infection in humans.

Src family kinase activity drives cytomegalovirus reactivation by recruiting MOZ histone acetyltransferase activity to the viral promoter

Human cytomegalovirus (HCMV) latency and reactivation rely on a complex interplay between cellular differentiation, cell signaling pathways, and viral gene functions. HCMV reactivation in dendritic cells (DCs) is triggered by IL-6 and extracellular signal-regulated kinase (ERK)–mitogen-activated protein kinase signaling. However, activation of the same pathway fails to reactivate HCMV in other myeloid cell types, despite this signaling axis being active in those cells. We hypothesized that IL-6–induced ERK activation initiates the changes in chromatin structure required for viral reactivation but that a concomitant signal is necessary to complete the changes in chromatin structure required for gene expression to occur. Using a differential phosphoproteomics approach in cells that do or do not support IL-6–induced viral reactivation, we identified the concomitant activation of an Src family kinase (SFK), hematopoietic cell kinase (HCK), specifically in DCs in response to IL-6. Pharmacological and genetic inhibition of HCK activity indicated that HCK is required for HCMV reactivation. Furthermore, the HCK/SFK activity was linked to recruitment of the monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase to the viral promoter, which promoted histone acetylation after ERK-mediated histone phosphorylation. Importantly, pharmacological and genetic inhibition of MOZ activity prevented reactivation. These results provide an explanation for the selective activation of viral gene expression in DCs by IL-6, dependent on concomitant SFK and ERK signaling. They also reveal a previously unreported role for SFK activity in the regulation of chromatin structure at promoters in eukaryotic cells via MOZ histone acetyltransferase activity.

Signal Transduction and Pathogenic Modifications at the Melanocortin-4 Receptor: A Structural Perspective.

The melanocortin-4 receptor (MC4R) can be endogenously activated by binding of melanocyte-stimulating hormones (MSH), which mediates anorexigenic effects. In contrast, the agouti-related peptide (AgRP) acts as an endogenous inverse agonist and suppresses ligand-independent basal signaling activity (orexigenic effects). Binding of ligands to MC4R leads to the activation of different G-protein subtypes or arrestin and concomitant signaling pathways. This receptor is a key protein in the hypothalamic regulation of food intake and energy expenditure and naturally-occurring inactivating MC4R variants are the most frequent cause of monogenic obesity. In general, obesity is a growing problem on a global scale and is of social, medical, and economic relevance. A significant goal is to develop optimized pharmacological tools targeting MC4R without adverse effects. To date, this has not been achieved because of inter alia non-selective ligands across the five functionally different MCR subtypes (MC1-5R). This motivates further investigation of (i) the three-dimensional MC4R structure, (ii) binding mechanisms of various ligands, and (iii) the molecular transfer process of signal transduction, with the aim of understanding how structural features are linked with functional-physiological aspects. Unfortunately, experimentally elucidated structural information is not yet available for the MC receptors, a group of class A G-protein coupled receptors (GPCRs). We, therefore, generated MC4R homology models and complexes with interacting partners to describe approximate structural properties associated with signaling mechanisms. In addition, molecular insights from pathogenic mutations were incorporated to discriminate more precisely their individual malfunction of the signal transfer mechanism.

Demyelination-remyelination in the Central Nervous System: Ligand-dependent Participation of the Notch Signaling Pathway.

Multiple sclerosis (MS) is an immune-mediated central nervous system disease mostly affecting young people. Multiple sclerosis and other neurodegenerative and white matter disorders involve oligodendrocyte (OL) damage and demyelination. Therefore, elucidating the signaling pathways involved in the remyelination process through the maturation of OL progenitor cells (OPCs) may contribute to the development of new therapeutic approaches. In this context, this paper further characterizes toxic cuprizone (CPZ)-induced demyelination and spontaneous remyelination in rats and investigates the role of ligand-dependent Notch signaling activation along demyelination/remyelination both in vivo and in vitro. Toxic treatment generated an inflammatory response characterized by both microgliosis and astrogliosis. Interestingly, early demyelination revealed an increase in the proportion of Jagged1+/GFAP+ cells, which correlated with an increase in Jagged1 transcript and concomitant Jagged1-driven Notch signaling activation, particularly in NG2+ OPCs, in both the corpus callosum (CC) and subventricular zone (SVZ). The onset of remyelination then exhibited an increase in the proportion of F3/contactin+/NG2+ cells, which correlated with an increase in F3/contactin transcript during ongoing remyelination in the CC. Moreover, neurosphere cultures revealed that neural progenitor cells present in the brain SVZ of CPZ-treated rats recapitulate in vitro the mechanisms underlying the response to toxic injury observed in vivo, compensating for mature OL loss. Altogether, the present results offer strong evidence of cell-type and ligand-specific Notch signaling activation and its time- and area-dependent participation in toxic demyelination and spontaneous remyelination.

Combined blockade of MEK and PI3KCA as an effective antitumor strategy in HER2 gene amplified human colorectal cancer models

BackgroundTargeting the epidermal growth factor receptor (EGFR) either alone or in combination with chemotherapy is an effective treatment for patients with RAS wild-type metastatic colorectal cancer (mCRC). However, only a small percentage of mCRC patients receive clinical benefits from anti-EGFR therapies, due to the development of resistance mechanisms. In this regard, HER2 has emerged as an actionable target in the treatment of mCRC patients with resistance to anti-EGFR therapy.MethodsWe have used SW48 and LIM1215 human colon cancer cell lines, quadruple wild-type for KRAS, NRAS, BRAF and PI3KCA genes, and their HER2–amplified (LIM1215-HER2 and SW48-HER2) derived cells to perform in vitro and in vivo studies in order to identify novel therapeutic strategies in HER2 gene amplified human colorectal cancer.ResultsLIM1215-HER2 and SW48-HER2 cells showed over-expression and activation of the HER family receptors and concomitant intracellular downstream signaling including the pro-survival PI3KCA/AKT and the mitogenic RAS/RAF/MEK/MAPK pathways. HER2-amplified cells were treated with several agents including anti-EGFR antibodies (cetuximab, SYM004 and MM151); anti-HER2 (trastuzumab, pertuzumab and lapatinib) inhibitors; anti-HER3 (duligotuzumab) inhibitors; and MEK and PI3KCA inhibitors, such as refametinib and pictilisib, as single agents and in combination. Subsequently, different in vivo experiments have been performed. MEK plus PI3KCA inhibitors treatment determined the best antitumor activity. These results were validated in vivo in HER2-amplified patient derived tumor xenografts from three metastatic colorectal cancer patients.ConclusionsThese results suggest that combined therapy with MEK and PI3KCA inhibitors could represent a novel and effective treatment option for HER2-amplified colorectal cancer.