Multi-Modal Exercise Training and Protein-Pacing Enhances Physical Performance Adaptations Independent of Growth Hormone and BDNF but May Be Dependent on IGF-1 in Exercise-Trained Men

OBJECTIVE: Protein-pacing (P; 5-6meals/day @ 2.0g/kgBW/day) and multi-mode exercise (RISE; resistance, interval, stretching, endurance) training (PRISE) improves muscular endurance, strength, power and arterial health in exercise-trained women. The current study extends these findings by examining PRISE on fitness, growth hormone (GH), insulin-like growth factor-1 (IGF-1), and brain-derived neurotrophic factor (BDNF) response, cardiometabolic health, and body composition in exercise-trained men. DESIGN: Twenty active males (\u3e4daysexercise/week) completed either: PRISE (n=11) or RISE (5-6meals/day @ 1.0g/kgBW/day; n=9) for 12weeks. Muscular strength (1-repetition maximum bench and leg press, 1-RM BP, and 1-RM LP), endurance (sit-ups, SU; push-ups, PU), power (squat jump, SJ, and bench throw, BT), flexibility (sit-and-reach, SR), aerobic performance (5km cycling time-trial, TT), GH, IGF-1, BDNF, augmentation index, (AIx), and body composition, were assessed at weeks 0 (pre) and 13 (post). RESULTS:At baseline, no differences existed between groups except for GH (RISE, 230±13 vs. PRISE, 382±59pg/ml, p CONCLUSIONS: Exercise-trained men consuming a P diet combined with multi-component exercise training (PRISE) enhance muscular power, strength, aerobic performance, and flexibility which are not likely related to GH or BDNF but possibly to IGF-1 response

Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses

<div><p>Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005324#ppat.1005324.ref001" target="_blank">1</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005324#ppat.1005324.ref004" target="_blank">4</a>]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses.</p></div

G10-mediated Induction of mRNA in PBMCs.

<p>mRNA synthesis of indicated genes in human peripheral blood mononuclear cells (PBMC) following 8h exposure to indicated concentration of G10 (A) or ppp-dsRNA (12.5μg/mL) or 2’3’-cGAMP (28μM) (B). Indicated values represent average mRNA fold change ±SD from duplicate experiments relative to cells exposed to 1% DMSO.</p

IRF3 is Required for G10-Dependent Transcription and Anti-Alphaviral Activity.

<p>(A) Immunoblot showing IRF3, STAT1, and GAPDH in THF-ISRE stably transduced with Cas9 and CRISPR gRNA directed against either STAT1 (THF-ISRE-ΔSTAT1) or IRF3 (THF-ISRE-ΔIRF3) as indicated. (B) Induction of IRF3/IFN-dependent LUC in THF lacking STAT1 following 7h exposure to 100μM G10, UV-inactivated CMV, or 1000U/mL IFNβ. Values displayed are average fold changes ±SD of quadruplicate measurements relative to cells exposed only to 1% DMSO. (C) Induction of IRF3/IFN-dependent LUC in THF lacking IRF3 following 7h exposure to 100μM G10, UV-inactivated CMV, or 1000U/mL IFNβ. Values displayed are as in B. (D) Immunoblot of lysates from THF-ISRE following 6h exposure to DMSO, UV-CMV, SeV or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, and GAPDH. (E) Average media titers +SD of CHIKV, VEEV, and SINV at 24 (VEEV) or 48hpi (CHIKV, SINV) obtained from THF-ISRE-ΔIRF3 cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

G10 Elicits IRF3 phosphorylation and Anti-Alphaviral Activity in Cells Lacking IPS1.

<p>(A) Immunoblot of lysates from THF-ISRE-ΔIPS1 following 6h exposure to DMSO, UV-CMV, SeV or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, IPS1, STING, and GAPDH. (B) Average media titers +SD of CHIKV, VEEV, and SINV at 24h (VEEV) or 48h (CHIKV, SINV) post infection obtained from THF-ISRE-ΔIPS1 cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p

G10 Induces IFN/IRF3- but not NF-κB-Dependent Transcription in Human Fibroblasts.

<p>(A) Dose-dependent expression of IRF3/IFN-dependent luciferase (LUC) in telomerized human fibroblasts (THF). Values displayed are average fold changes ±SD of quadruplicate measurements of luminescence following 7h exposure to indicated concentration of G10 relative to cells exposed only to 1% DMSO (all samples normalized to 1% DMSO). (B) mRNA transcription of genes dependent on IRF3/IFN following 7h exposure to 100μM G10 or UV-CMV. Indicated values represent average ±SEM mRNA fold change relative to cells exposed to 1% DMSO from duplicate experiments. (C) Induction of NF-κB-dependent LUC signal in THF reporter cells following 7h exposure to 1μg/mL LPS, 160 HA units/mL SeV, 1ng/mL TNFα or indicated concentration of G10. Values displayed are as described in (A). (D) mRNA transcription of of NF-κB-dependent genes following 7h exposure to 100μM G10 or 160 HA units/mL SeV. Indicated values are mRNA fold change relative to cells exposed to 1% DMSO and are representative of duplicate experiments.</p

Comparative Kinetics and Dose-Dependence of Innate Immune Activation by G10 and 2’3’-cGAMP.

<p>(A) Immunoblot of lysates from THF-ISRE cells following exposure to G10 (100μM) or transfected 2’3’-cGAMP (42.3μM) for indicated time showing phosphorylation status of IRF3 S386, total IRF3, and GAPDH. (B) mRNA synthesis of indicated genes in THF following 8h exposure to indicated concentration of G10 (blue) or 2’3’-cGAMP (red). Indicated values represent average mRNA fold change ±SD from duplicate experiments relative to cells exposed to 1% DMSO.</p

G10-Mediated IRF3 Phosphorylation, ISG mRNA Induction, and Anti-Alphaviral Activity are not Detectable in Cells Lacking STING.

<p>(A) Immunoblot of lysates from THF-ΔSTING following 7h exposure to 1% DMSO, 0.1μg/mL poly(I:C), SeV, UV-CMV, 1μg/mL 2’3’-cGAMP, or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, IPS1, STING, and GAPDH. (B) Expression of IRF3/IFN-dependent LUC in THF-ISRE-ΔIPS1 and THF-ISRE-ΔSTING following 7h exposure to 1% DMSO, indicated concentrations of G10, SeV, UV-CMV, or 1μg/mL LPS. Values are presented as average fold change in quadruplicate measurements ±SD relative to cells treated with 1% DMSO. (C) Average fold changes ±SD from duplicate experiments of ISG54, ISG15, and Viperin mRNA relative to cells treated with 1% DMSO in THF-ISRE-ΔIPS1 (black bars) or THF-ISRE-ΔSTING (gray bars) following exposure to UV-CMV, SeV, or 100μM G10. (D) Media titers of CHIKV and VEEV at 24h (VEEV) or 48h (CHIKV) obtained from THF-ISRE-ΔSTING cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.</p