Introduction {#Sec1}
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Regular exercise may reduce the risk of major vascular thrombotic events and protect people against cardiovascular diseases^[@CR1],\ [@CR2]^. Moreover, high-intensity exercise elicits a greater central hemodynamic adaptation than low and moderate levels of exercise^[@CR3]^. However physical exercise imposes, paradoxically, both enhancing and suppressing effects on platelet reactivity and coagulation, depending on the type and intensity of exercise^[@CR1]^. Systemic hypoxia has detrimental effects on the haemostatic system, such as accelerating prothrombinase complex assembly and facilitating thrombin generation (TG)^[@CR4],\ [@CR5]^. Pathological investigations have also demonstrated that a hypercoagulable state contributes to increased risks of vascular thrombotic events in patients with episodic hypoxia, such as obstructive sleep apnea^[@CR6]^ and chronic obstructive pulmonary disease^[@CR7]^. To the best of our knowledge, what kind of exercise strategy improves aerobic capacity and simultaneously increases the resistance to thrombotic risk provoked by hypoxia has not yet been established.

Increased thrombin activity is an essential pathogenic process of cardiovascular diseases^[@CR8]^. Functional mitochondria in platelet has demonstrated the direct involvements of the cellular ATP production, redox balance, and thrombogenesis^[@CR9],\ [@CR10]^. Platelet activation results in its mitochondrial membrane potential collapse and reactive oxygen species (ROS) production, consequently facilitating the exposure of phosphatidylserine (PS)^[@CR10]^. The negatively charged PS on platelets can bind coagulant factors VIII (FVIII), Va, and Xa, providing a strongly catalytic surface for the assembly of prothrombinase and tenase, thus triggering TG in blood^[@CR10]^. Our early study further demonstrated that severe hypoxic exposure (12% O~2~) promoted FVIII-dependent TG by elevating oxidative stress; this hypoxic effect was ameliorated by pretreatment with antioxidant vitamin E^5^. However, a previous study has indicated that exercise training decreased the coagulant factor expression and PS exposure of platelets and the shedding of procoagulant microparticles from platelets, thereby depressing dynamic TG in platelets^[@CR11]^.

Warm-up exercise (40%VO~2max~) has been shown to reduce high-intensity exercise (80%VO~2max~)-induced risks of inflammatory thrombosis associated with leukocytes and platelets, which is a form of preconditioning^[@CR12]^. Recently, our investigation revealed that high-intensity interval exercise training (HIT) that consists of alternating mild-(40%VO~2max~) and high-(80%VO~2max~) intensity exercise significantly suppressed neutrophil-promoted blood coagulation by down-regulating expression of procoagulant factors under hypoxic condition^[@CR13]^. Accordingly, we further hypothesize that HIT effectively reduces platelet-induced TG undergoing hypoxic stress, which is associated with alleviating platelet mitochondrial dysfunction cause by hypoxia.

To answer the abovementioned questions, this study evaluated how two isovolumic exercise regimens \[*i*.*e*., HIT (3-minute intervals at 40% and 80%VO~2max~) and moderate-intensity continuous exercise training (MCT, sustained 60%VO~2max~)\] for 6 weeks affected (i) mitochondrial oxidative phosphorylation (OXPHOS) and oxidative stress, (ii) mitochondrial biogenesis, and (iii) dynamic TG in platelets following hypoxic exercise (HE, 100 W under 12% O~2~ for 30 min). The aim of the present study is to establish an effective exercise strategy for improving individual aerobic capacity and simultaneously ameliorating the risk of platelet mitochondrial dysfunction and subsequent TG evoked by hypoxic stress.

Results {#Sec2}
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Aerobic fitness {#Sec3}
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Anthropometric variables did not significantly differ among the three groups at the beginning of the study (Table [1](#Tab1){ref-type="table"}). Both HIT and MCT for 6 weeks lowered HR and systolic blood pressure at rest, as well as, increased work-rate, minute ventilation (V~E~), O~2~ consumption (VO~2~), and CO~2~ production (VCO~2~) at the ventilation threshold and peak performance (Table [1](#Tab1){ref-type="table"}, *P* \< 0.05). Moreover, the HIT group exhibited a greater improvement in pulmonary ventilation and aerobic capacity than did the MCT group (Table [1](#Tab1){ref-type="table"}, *P* \< 0.05). However, control subjects that did not receive exercise intervention (CTL) for 6 weeks showed no changes in these cardiopulmonary responses to a graded exercise test (GXT) (Table [1](#Tab1){ref-type="table"}).Table 1The effects of interval and continuous exercise regimens on exercise performance.HITMCTCTLPrePostPrePostPrePost***Anthropometric parameters***Age (year)22.2 ± 0.4---22.1 ± 0.5---21.9 ± 0.7---Height (cm)171.0 ± 1.0---172.1 ± 1.2---172.2 ± 1.3---Weight (kg)69.7 ± 1.170.3 ± 2.167.3 ± 1.967.0 ± 1.867.8 ± 3.167.8 ± 3.1BMI (kg/m^2^)23.8 ± 0.523.4 ± 0.622.0 ± 0.521.9 ± 0.522.3 ± 0.822.3 ± 0.8HR (beats/min)72 ± 168 ± 2^+^71 ± 267 ± 2^+^73 ± 272 ± 3SBP (mmHg)120 ± 2115 ± 2^+^121 ± 3116 ± 3^+^123 ± 2122 ± 3DBP (mmHg)74 ± 272 ± 274 ± 372 ± 275 ± 374 ± 3***Ventilation threshold***Work-rate (watt)100 ± 5156.5 ± 7.5\*^+^99.4 ± 5.7131.8 ± 6.0\*104.0 ± 6.2108.1 ± 6.2HR (beats/min)133 ± 2154 ± 2.8\*^+^134 ± 3146 ± 4.0\*132 ± 4134 ± 5$\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$ ~E~ (l/min)36.2 ± 1.853.5 ± 3.0\*^+^36.5 ± 2.147.0 ± 2.6\*35.2 ± 4.236.5 ± 2.6$\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$O~2~ (ml/min/kg)16.4 ± 0.723.8 ± 1.0\*^+^17.5 ± 0.721.2 ± 0.9\*16.9 ± 0.917.0 ± 0.8$\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$CO~2~ (ml/min/kg)16.5 ± 0.723.8 ± 1.0\*^+^17.5 ± 0.721.4 ± 1.1\*17.0 ± 0.917.2 ± 0.9***Peak performance***Work-rate (watt)184.7 ± 6.3237.6 ± 7.0\*^+^182.4 ± 5.4213.5 ± 5.5\*180 ± 7182 ± 9HR (beats/min)194 ± 2195 ± 2193 ± 2194 ± 2195 ± 3194 ± 2$\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$ ~E~ (l/min)103.3 ± 3.0133.6 ± 4.2\*^+^98.7 ± 3.5114.1 ± 3.2\*105.1 ± 4.1104.2 ± 5.1$\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$O~2~ (ml/min/kg)31.7 ± 0.539.7 ± 0.6\*^+^32.2 ± 0.636.2 ± 0.6\*31.4 ± 0.631.4 ± 0.6$\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$CO~2~ (ml/min/kg)38.0 ± 0.647.4 ± 0.7^\*,+^38.6 ± 0.843.4 ± 0.8\*37.7 ± 0.737.5 ± 0.6Values were mean ± SEM. HIT, high-intensity interval training group; MCT, moderate-intensity continuous training group; CTL, control group; Pre, pre-intervention; Post, post-intervention; BMI, body mass index; $\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$ ~E~, minute ventilation; $\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$O~2~, oxygen consumption; $\documentclass[12pt]{minimal}
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                \begin{document}$$\dot{{\rm{V}}}$$\end{document}$CO~2~, carbon dioxide production. \**P* \< 0.05, Pre vs. Post; ^+^ *P* \< 0.05, HIT vs. MCT.

Mitochondrial membrane potential (MP), matrix oxidant burden (MOB), and biogenesis in platelets {#Sec4}
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Acute bout of 12% O~2~ exercise significantly increased platelet counts in blood (*P* \< 0.05, Table [2](#Tab2){ref-type="table"}). However, blood platelet counts before or after the HE test remained unchanged following the 6-week intervention with HIT, MCT or CTL (Table [2](#Tab2){ref-type="table"}). Figure [1](#Fig1){ref-type="fig"} shows the analysis of platelet mitochondrial count (Fig. [1e--h](#Fig1){ref-type="fig"}), MP (Fig. [1i--l](#Fig1){ref-type="fig"}), and MOB (Fig. [1m--p](#Fig1){ref-type="fig"}) using a FACScan flow cytometer. Although no changes were observed in the mitochondrial count (Fig. [2a--c](#Fig2){ref-type="fig"}), the HE decreased the mitochondrial MP (Fig. [2d--f](#Fig2){ref-type="fig"}, *P* \< 0.05) and elevated the MOB (Fig. [2g--i](#Fig2){ref-type="fig"}, *P* \< 0.05) in platelets. After 6 weeks of the intervention, HIT, rather than MCT, inhibited the HE-induced decrease of mitochondrial MP (Fig. [2d](#Fig2){ref-type="fig"}, *P* \< 0.05) and increase of MOB (Fig. [2g](#Fig2){ref-type="fig"}, *P* \< 0.05) in platelets. However, no significant changes in the mitochondrial count, MP, and MOB of platelets were observed after CTL for 6 weeks (Fig. [2c,f,i](#Fig2){ref-type="fig"}).Table 2The effects of interval and continuous exercise regimens on platelet count and mitochondrial biogenesis.HITMCTCTLPrePostPrePostPrePost***Platelet count (x10*** ^***3***^ ***cells/μl)***R224 ± 11216 ± 12232 ± 10226 ± 18234 ± 12237 ± 9HE264 ± 21\*252 ± 23\*272 ± 16\*266 ± 12\*284 ± 20\*287 ± 17\****Platelet mitochondrial biogenesis (mean fluorescence intensity)***Complex IIR21.7 ± 2.822.1 ± 2.422.6 ± 1.922.5 ± 2.623.5 ± 1.923.4 ± 2.5HE21.6 ± 2.323.1 ± 2.823.0 ± 1.623.9 ± 2.523.7 ± 2.124.3 ± 1.7Complex IVR16.9 ± 2.817.0 ± 2.516.3 ± 1.716.9 ± 2.117.4 ± 1.316.9 ± 1.5HE16.0 ± 2.717.1 ± 1.916.7 ± 1.116.8 ± 1.416.9 ± 1.717.0 ± 0.8Ratio of Complex IV to Complex IIR0.78 ± 0.040.77 ± 0.030.72 ± 0.050.71 ± 0.040.74 ± 0.040.72 ± 0.03HE0.74 ± 0.060.73 ± 0.040.73 ± 0.040.72 ± 0.050.71 ± 0.030.70 ± 0.04Values were mean ± SEM. HIT, high-intensity interval training group; MCT, moderate-intensity continuous training group; CTL, control group; Pre, pre-intervention; Post, post-intervention; R, resting; HE, hypoxic exercise test. \**P* \< 0.05, R vs. HE. Figure 1Graph showing the effect of high-intensity interval training (**HIT**) on mitochondrial content, mitochondrial membrane potential, and matrix oxidant burden in platelets using a FACScan flow cytometer. **Pre**, before HIT; **Post**, after HIT; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test. **Blank (a--d)**, platelets without treating fluorescent dye; **Mitochondrial content (e--h)**, platelets stained with MitoTracker Green FM; **Mitochondrial membrane potential (i--l)**, platelet stained with tetramethylrhodamine ethyl ester; **Matrix oxidant burden (m--p)**, platelet stained with MitoSOX Red; **MFI**, mean fluorescence intensity. Figure 2Effects of interval and continuous exercise regimens on **(a--c)** mitochondrial content, **(d--f)** mitochondrial membrane potential, and **(g--i**) matrix oxidant burden in platelets. **HIT**, high-intensity interval training group **(a**,**d**,**g)**; **MCT**, moderate-intensity continuous training **(b**,**e**,**h)**; **CTL**, control group **(c**,**f**,**i)**; **Pre**, pre-intervention; **Post**, post-intervention; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test; **MFI**, mean fluorescence intensity. **\*** *P* \< 0.05, **R** vs. **HE**; ^+^ *P* \< 0.05, **Pre** vs. **Post**. Values were mean ± SEM.

In mitochondrial biogenesis, acute HE did not change the levels of Complex IV and II, as well as, the ratio of Complex IV to II in platelets (Table [2](#Tab2){ref-type="table"}). Furthermore, no significant changes in these mitochondrial biogenetic variables of platelets occurred after 6-week interventions with HIT, MCT, or CTL (Table [2](#Tab2){ref-type="table"}).

Mitochondrial respiration of platelets {#Sec5}
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Figures [3a--h](#Fig3){ref-type="fig"} show the analysis of O~2~ consumption rate (OCR) in the intact (3a--3d) and permeabilized (3e--3h) platelets using high-resolution respirometry (Oroboros O2K). An acute bout of 12%O~2~ exercise significantly decreased ATP-linked OCR (Fig. [4a--c](#Fig4){ref-type="fig"}, *P* \< 0.05) and the reserve capacity of OCR (Fig. [4g--i](#Fig4){ref-type="fig"}, *P* \< 0.05) in intact platelets. The HE also suppressed the fatty acid oxidation (FAO)- (Fig. [5d--f](#Fig5){ref-type="fig"}, *P* \< 0.05), Complex I- (Fig. [5g--i](#Fig5){ref-type="fig"}, *P* \< 0.05), and Complex II- (Fig. [5j--l](#Fig5){ref-type="fig"}, *P* \< 0.05) mediated OCRs in permeabilized platelets. After 6 weeks, both HIT and MCT significantly elevated the reserve capacity of OCR in intact platelets at rest (Fig. [4g and h](#Fig4){ref-type="fig"}, *P* \< 0.05), whereas only HIT alleviated the suppression of the OCR reserve capacity caused by HE (Fig. [4g](#Fig4){ref-type="fig"}, *P* \< 0.05). Moreover, the two exercise regimens enhanced the Complex II-mediated OCR at rest (Fig. [5j and k](#Fig5){ref-type="fig"}, *P* \< 0.05), while only HIT diminished the depression of FAO- (Fig. [5d](#Fig5){ref-type="fig"}), Complex I- (Fig. [5g](#Fig5){ref-type="fig"}), and Complex II- (Fig. [5j](#Fig5){ref-type="fig"}) mediated OCRs and the capacity for electron transport system (ETS) (Fig. [5p](#Fig5){ref-type="fig"}) in platelets caused by HE. However, there were no significant changes in the HE-mediated mitochondrial respiration of platelets after 6 weeks of CTL (Figs [4](#Fig4){ref-type="fig"} and [5](#Fig5){ref-type="fig"}).Figure 3Graph showing measurement of mitochondrial O~2~ consumption rate (OCR) in platelets using a high-resolution respirometry (Oroboros O2K). **(a--d)**, the OCR protocol in intact platelets; **(e--h)**, the OCR \[substrate, uncoupler, inhibitor titration (SUIT)\] protocol in permeabilized platelets. **Omy**, oligomycin; **FCCP**, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; **Rot**, rotenone; **AA**, antimycin A; **M**, malate; **C**, palmitoyl-DL carnitine-HCl; **Dig**, digitonin; **ADP**, adenosine diphosphate; **P**, pyruvate; **G**, glutamate; **S**, succinate; **Cyst C**, cytochrome c; **MA**, malonic acid; **Mt**, myxothiazol. **Pre**, before high-intensity interval training (HIT); **Post**, after HIT; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test. Figure 4Effects of interval and continuous exercise regimens on mitochondrial O~2~ consumption rate (OCR) in the intact platelets. **HIT (a**,**d**,**g**,**j**,**m)**, high-intensity interval training group; **MCT (b**,**e**,**h**,**k**, **n)**, moderate-intensity continuous training; **CTL (c**,**f**,**i**,**l**,**o)**, control group; **Pre**, pre-intervention; **Post**, post-intervention; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test; **ATP**, ATP-linked OCR; **Uncouple**, uncouple OCR; **RESERVE**, the reserve capacity of OCR; **ROX**, residual O~2~ consumption. **\*** *P* \< 0.05, **R** vs. **HE**; ^+^ *P* \< 0.05, **Pre** vs. **Post**. Values were mean ± SEM. Figure 5Effects of interval and continuous exercise regimens on mitochondrial O~2~ consumption rate (OCR) in the permeabilized platelets. **HIT (a**,**d**,**g**,**j**,**m**,**p**,**s)**, high-intensity interval training group; **MCT (b**,**e**,**h**,**k**,**n**,**q**,**t)**, moderate-intensity continuous training; **CTL (c**,**f**,**i**,**l**,**o**,**r**,**u)**, control group; **Pre**, pre-intervention; **Post**, post-intervention; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test; **ATP**, ATP-linked OCR; **FAO**, fatty acid oxidation; **ETS**, electron transport system; **CI**, Complex I respiration; **CII**, Complex I respiration; **ROX**, Residual O~2~ consumption. **\*** *P* \< 0.05, **Rt** vs. **HE**; ^+^ *P* \< 0.05, **Pre** vs. **Post**. Values were mean ± SEM.

Before various interventions, the HE test also suppressed the bioenergetics health index (BHI) value in platelets (Fig. [6a--c](#Fig6){ref-type="fig"}, *P* \< 0.05). After 6 weeks, both HIT (Fig. [6a](#Fig6){ref-type="fig"}, *P* \< 0.05) and MCT (Fig. [6b](#Fig6){ref-type="fig"}, *P* \< 0.05) significantly elevated the BHI value in platelets at rest, whereas only HIT attenuated the suppression of BHI caused by HE (Fig. [6a](#Fig6){ref-type="fig"}, *P* \< 0.05). However, the activities of Complex IV in platelet mitochondria at rest or after HE remained unchanged following 6 weeks of HIT, MCT, or CTL (Fig. [6d--f](#Fig6){ref-type="fig"}).Figure 6Effects of interval and continuous exercise regimens on **(a--c**) bioenergetics health index (BHI) and **(d--f)** Complex IV activity in platelets. **HIT**, high-intensity interval training group **(a**,**d)**; **MCT**, moderate-intensity continuous training **(b**,**e)**; **CTL**, control group **(c**,**f)**; **Pre**, pre-intervention; **Post**, post-intervention; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test. **\*** *P* \< 0.05, **R** vs. **HE**; ^+^ *P* \< 0.05, **Pre** vs. **Post**. Values were mean ± SEM.

Enzyme activities of glycolysis and the Krebs cycle in platelets {#Sec6}
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Acute HE increased the activity of lactate dehydrogenase (LDH) and decreased the activities of citrate synthase (CS) and succinate dehydrogenase (SDH) in platelets (Table [3](#Tab3){ref-type="table"}, *P* \< 0.05). After 6 weeks of the intervention, HIT, but not MCT, attenuated the extents of the HE-enhanced LDH activity and the HE-depressed CS and SDH activities in platelets (Table [3](#Tab3){ref-type="table"}). However, the two exercise regimens did not influence the activities of hexokinase (HK) and pyruvate dehydrogenase (PDH) in platelets at rest and after HE (Table [3](#Tab3){ref-type="table"}). Additionally, various enzyme activities of glycolysis and Krebs cycle in platelets at rest or after HE remained unchanged after 6 weeks of CTL (Table [3](#Tab3){ref-type="table"}).Table 3The effects of interval and continuous exercise regimens on enzyme activities of glycolysis and Krebs cycle in platelets.HITMCTCTLPrePostPrePostPrePost***Glycolysis***Hexokinase activity (nmol/min/10^8^ cells)   R27.9 ± 1.926.7 ± 1.724.7 ± 2.425.9 ± 1.124.4 ± 1.423.3 ± 1.3   HE27.5 ± 1.727.3 ± 1.124.8 ± 2.726.6 ± 1.525.7 ± 1.223.8 ± 1.1Pyruvate dehydrogenase activity (nmol/min/10^8^ cells)   R1.18 ± 0.031.13 ± 0.031.16 ± 0.031.04 ± 0.031.26 ± 0.051.18 ± 0.04   HE1.30 ± 0.041.17 ± 0.031.27 ± 0.041.12 ± 0.031.26 ± 0.061.26 ± 0.05Lactate dehydrogenase activity (nmol/min/10^8^ cells)   R32.2 ± 4.530.1 ± 1.631.6 ± 2.531.8 ± 2.535.6 ± 2.433.5 ± 2.6   HE40.1 ± 4.6\*33.6 ± 1.5^+^41.2 ± 2.4\*37.9 ± 2.5\*42.5 ± 2.2\*41.6 ± 2.8\****Krebs cycle***Citrate synthase activity (nmol/min/10^8^ cells)   R5.50 ± 0.225.83 ± 0.255.63 ± 0.175.74 ± 0.215.54 ± 0.175.68 ± 0.34   HE4.45 ± 0.32\*5.88 ± 0.43^+^4.61 ± 0.24\*4.61 ± 0.32\*4.52 ± 0.24\*4.55 ± 0.29\*Succinate dehydrogenase activity (nmol/min/10^8^ cells)   R9.46 ± 0.829.59 ± 1.329.37 ± 0.919.62 ± 0.839.12 ± 0.429.41 ± 0.45   HE6.77 ± 0.91\*8.82 ± 0.81^+^6.39 ± 0.92\*6.83 ± 0.72\*6.47 ± 0.61\*6.57 ± 0.60\*Values were mean ± SEM. HIT, high-intensity interval training group; MCT, moderate-intensity continuous training group; CTL, control group; Pre, pre-intervention; Post, post-intervention; R, resting; HE, hypoxic exercise test. \**P* \< 0.05, R vs. HE; ^+^ *P* \< 0.05, *P*re vs. Post.

Dynamic TG in platelet-rich plasma (PRP) {#Sec7}
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Figure [7a--g](#Fig7){ref-type="fig"} show the samples of the HE-mediated dynamic TG in pretreating PRP with various mitochondrial modulators following 6 weeks of HIT. With respect to the analytic parameters of dynamic TG, acute HE increased the ETP (Table [3](#Tab3){ref-type="table"}, *P* \< 0.05), peak height (Table [4](#Tab4){ref-type="table"}, *P* \< 0.05), and rate (Fig. [8a--c](#Fig8){ref-type="fig"}, *P* \< 0.05) of TG but not changed the lag time of TG (Table [4](#Tab4){ref-type="table"}) in PRP before the intervention. Moreover, the changes of parameters of dynamic TG caused by HE were attenuated by pretreating PRP with oligomycin (Table [4](#Tab4){ref-type="table"} and Fig. [8d--f](#Fig8){ref-type="fig"}) or rotenone plus antimycin A (Table [4](#Tab4){ref-type="table"} and Fig. [8j--l](#Fig8){ref-type="fig"}). Six weeks of HIT (Fig. [7a--d](#Fig7){ref-type="fig"}) and MCT (Fig. [7E--H](#Fig7){ref-type="fig"}) significantly decreased the extents of HE-promoted ETP (Table [4](#Tab4){ref-type="table"}, *P* \< 0.05) and peak height (Table [4](#Tab4){ref-type="table"}, *P* \< 0.05) and rate (Fig. [8a](#Fig8){ref-type="fig"}, *P* \< 0.05) of TG in PRP. However, pretreating PRP with oligomycin (Table [4](#Tab4){ref-type="table"}, Fig. [8d--f](#Fig8){ref-type="fig"}), rather than rotenone plus antimycin A (Table [4](#Tab4){ref-type="table"} and Fig. [8j--l](#Fig8){ref-type="fig"}), inhibited the training effects of HIT and MCT on the dynamic TG in PRP. Additionally, CLT did not significantly change the values of resting and HE-mediated dynamic TG parameters in untreated and oligomycin- or rotenone plus antimycin A-treated PRP (Table [4](#Tab4){ref-type="table"} and Fig. [8](#Fig8){ref-type="fig"}).Figure 7The calibrated, automatic thrombinographic analysis of high-intensity interval training (**HIT**) effect on dynamic thrombin generation in platelet-rich plasma (PRP). **Pre-HIT**, before HIT; **Post-HIT**, after HIT; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test; **Omy**, oligomycin; **FCCP**, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; **Rot**, rotenone; **AA**, antimycin A. Table 4The effects of interval and continuous exercise regimens on dynamic thrombin generation in platelet-rich plasma.HITMCTCTLPrePostPrePostPrePost***Vehicle***Lag time (min)   R9.1 ± 0.19.0 ± 0.19.1 ± 0.28.9 ± 0.19.1 ± 0.29.1 ± 0.2   HE9.1 ± 0.19.0 ± 0.19.2 ± 0.18.9 ± 0.19.2 ± 0.29.3 ± 0.2ETP (mM × min)   R879 ± 25876 ± 24873 ± 14871 ± 15855 ± 25885 ± 25   HE1105 ± 34\*845 ± 6^+^1101 ± 35\*870 ± 14^+^1103 ± 42\*1139 ± 48\*Peak height (mM)   R50.7 ± 1.745.5 ± 1.252.2 ± 1.349.6 ± 1.450.3 ± 3.751.0 ± 1.2   HE70.1 ± 3.2\*45.0 ± 1.3^+^67.6 ± 2.5\*49.1 ± 1.7^+^71.6 ± 5.1\*76.3 ± 2.9\****Oligomycin***Lag time (min)   R8.9 ± 0.19.0 ± 0.18.9 ± 0.18.8 ± 0.18.8 ± 0.28.7 ± 0.2   HE8.8 ± 0.18.9 ± 0.18.8 ± 0.18.7 ± 0.18.8 ± 0.28.9 ± 02ETP (mM × min)   R779 ± 31795 ± 17791 ± 17791 ± 14795 ± 25782 ± 20   HE825 ± 32788 ± 29845 ± 18803 ± 17846 ± 25834 ± 41Peak height (mM)   R45.0 ± 1.244.7 ± 1.645.3 ± 1.244.3 ± 1.644.2 ± 3.043.7 ± 1.2   HE47.0 ± 1.344.0 ± 1.546.8 ± 1.244.0 ± 1.446.2 ± 3.044.9 ± 1.0***Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone***Lag time (min)   R6.9 ± 0.27.0 ± 0.16.9 ± 0.27.0 ± 0.17.0 ± 0.36.9 ± 0.2   HE6.7 ± 0.27.0 ± 0.16.9 ± 0.26.9 ± 0.16.9 ± 0.27.1 ± 0.2ETP (mM × min)   R1005 ± 23955 ± 221027 ± 251032 ± 211069 ± 281040 ± 26   HE1222 ± 28\*951 ± 20^+^1215 ± 32\*1025 ± 18^+^1252 ± 30\*1243 ± 33\*Peak height (mM)   R78.1 ± 3.479.2 ± 2.982.6 ± 2.876.8 ± 2.983.6 ± 6.187.3 ± 2.1   HE108.1 ± 3.4\*78.6 ± 1.7^+^106.5 ± 2.7\*79.4 ± 2.62^+^111.7 ± 7.2\*112.5 ± 4.8\****Rotenone plus Antimycin A***Lag time (min)   R9.0 ± 0.29.0 ± 0.19.1 ± 0.28.8 ± 0.19.0 ± 0.28.7 ± 0.2   HE8.9 ± 0.28.9 ± 0.19.0 ± 0.18.8 ± 0.19.0 ± 0.28.8 ± 0.3ETP (mM × min)   R833 ± 22723 ± 21^+^840 ± 31749 ± 16^+^833 ± 20829 ± 26   HE847 ± 20735 ± 21^+^879 ± 27754 ± 13^+^859 ± 24841 ± 24Peak height (mM)   Rest49.0 ± 2.133.3 ± 0.9^+^45.3 ± 1.834.7 ± 1.3^+^46.4 ± 3.047.0 ± 1.3   HE50.4 ± 1.732.6 ± 1.0^+^48.1 ± 1.537.0 ± 1.8^+^49.8 ± 3.147.8 ± 1.5Values were mean ± SEM. HIT, high-intensity interval training group; MCT, moderate-intensity continuous training group; CTL, control group; Pre, pre-intervention; Post, post-intervention; R, resting; HE, hypoxic exercise test; ETP, endogenous thrombin potential. \**P* \< 0.05, R vs. HE; ^+^ *P* \< 0.05, *P*re vs. Post. Figure 8Effects of interval and continuous exercise regimens on the rate of thrombin generation in platelet-rich plasma. **HIT** (**a**,**d**,**g**,**j)**, high-intensity interval training group; **MCT (b**,**e**,**h**,**k**), moderate-intensity continuous training; **CTL** (**c**,**f**,**i**,**l)**, control group; **Pre**, pre-intervention; **Post**, post-intervention; **R**, resting; **HE**, hypoxic (12%O~2~) exercise test; **ETP**, endogenous thrombin potential; **Omy**, oligomycin; **FCCP**, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; **Rot**, rotenone; **AA**, antimycin A. **\*** *P* \< 0.05, **R** vs. **HE**; ^+^ *P* \< 0.05, **Pre** vs. **Post**. Values were mean ± SEM.

Discussion {#Sec8}
==========

Mitochondrial dysfunction is associated with oxidative stress and thought to be a common underlying mechanism of metabolic and cardiovascular diseases^[@CR14]--[@CR16]^. This study is the first to demonstrate that acute bout of 12% O~2~ exercise elevates mitochondrial oxidative stress and subsequently suppresses the CS and SDH activities and the ETS efficiency in platelets, which responses are accompanied by accelerating TG in PRP. Notably, the HIT regimen effectively ameliorates the HE-induced mitochondrial dysfunction of platelets by depressing the cellular oxidative stress, which may reduce the risk of platelet-mediated TG evoked by hypoxia.

Mitochondrial oxidative phosphorylation in platelets {#Sec9}
----------------------------------------------------

Mitochondria are highly sensitive to hypoxic stress and respond dynamically to changes in their cellular microenvironment^[@CR17]^. The present study showed that acute HE decreased mitochondrial MP and increased MOB in platelets, which may reflect mitochondrial oxidative damage of platelets caused by HE. Additionally, the HE-induced decrease in ATP-linked OCR and reserve capacity of OCR may be associated with modified activities of mitochondrial enzymes and/or an impeded the flow of electrons, thereby decreasing the ETS efficiency after HE. Acute HE also significantly decreased the CS and SDH activities and was accompanied by increased the LDH activity, suggesting that the HE shifts platelet metabolic pathways from mitochondrial respiration to glycolysis^[@CR18],\ [@CR19]^. Furthermore, the HE substantially depressed the capacity of Complex I- and II-related respirations and the efficiency of ETS in platelets in a substrate-rich environment. Summarily, acute HE globally decreases mitochondrial substrate availability and impairs mitochondrial bioenergetics and/or integrity in platelets.

In this investigation, the HIT regimen significantly enhanced platelet CS and SDH activities, as well as, Complex I- and II-related respirations following HE. The enhanced OCR of platelets by HIT may indicate a greater capacity of mitochondrial OXPHOS in platelets due to increased Complex protein activities of the ETS, elevated ATP production rate, or heightened levels of NADH and FADH~2~. Moreover, increased resistance to the HE-depressed platelet mitochondrial OCR after HIT may improve the flexibility of platelet activation caused by HE. A recent study has indicated that hypoxia or ischemia results in accumulation of intracellular succinate levels, thus leading to elevated mitochondrial ROS production^[@CR20]^. The elevation of oxidative stress may also induce succinate accumulation by decreasing SDH activity^[@CR21]^. Therefore, the increased SDH activity and Complex II respiration of platelets in the HIT groups may quickly eliminate succinate, thereby further retarding the ROS production from platelet mitochondria following HE.

Mitochondrial biogenesis in platelets {#Sec10}
-------------------------------------

Early studies on exercise intervention predominantly focused on mitochondrial functionality in skeletal muscles^[@CR22]^. An acute bout of exercise promotes transcriptional or post-translational regulation of peroxisome proliferator-activated receptor gamma coactivator-1α, whereas chronic exercise increases the rates of muscular mtDNA gene expression by upregulating Tfam in skeletal muscles^[@CR22]^. In the present study, no significant changes in resting and HE-related platelet biogenetic parameters, such as the Complex IV/II ratio, were observed after 6 weeks of HIT or MCT. Accordingly, we propose that platelet metabolic adaptation induced by HIT may be associated with improved ETS efficiency rather than modulated mitochondrial biogenesis in platelets.

Dynamic TG in PRP {#Sec11}
-----------------

Extremely hypoxic environments are associated with increased incidence of vascular thromboemoblic events^[@CR23],\ [@CR24]^. Blood is subjected to oxidative stress during extremely hypoxic exposure^[@CR4]^; elevated oxidative stress may contribute to coagulation system^[@CR4]^ and platelet activation^[@CR11]^. In the present study, acute HE facilitated TG in PRP and was accompanied by lost mitochondrial MP and increased MOB in platelets. Hence, increased ROS level by changed coupling of mitochondrial ETS may participate in platelet-mediated TG undergoing hypoxic stress. However, HIT for 6 week markedly depresses the HE-induced oxidative damage of platelet mitochondria and consequently attenuates the platelet-mediated TG caused by HE.

Limitations of study {#Sec12}
--------------------

Our small size in each group (n = 15) is a major limitation of this study. However, the results of aerobic capacity and mitochondrial bioenergetics and dynamic TG in platelets obtained from this investigation have high values of statistical power from 0.853 to 1.000. Additionally, the subjects used tended to be young, males, and healthy, thus further clinical evidence was required to extrapolate the present results to the elderly, females or patients with platelet or thrombotic disorders.

In this study, acute bout of HE promoted dynamic TG in PRP, whereas pretreating PRP with mitochondrial ETS inhibitors attenuated acute exercise effects. Moreover, 6 weeks of HIT also suppressed the extent of HE-promoted dynamic TG in PRP. Whether the suppression of HE-induced TG in PRP caused by HIT involves (i) change of plasma composition or/and (ii) down-regulation of procoagulant factors on platelets to diminish the enhancement of plasma TG by HE remains unresolved. Hence, the potential confounding effects of the different plasma compositions at various experimental conditions need further study.

Aim of this study mainly focus on examining the effects of exercise on platelet-induced TG rather than platelet reactivity (adhesion and aggregation). Our previous studies have investigated the effect of exercise training on platelet adhesiveness and aggregability and their underling mechanisms^[@CR25]^. However, the role of platelet mitochondrial function on exercise-mediated platelet reactivity requires further explored.

Conclusions {#Sec13}
-----------

Acute 12% O~2~ exercise elevated oxidative stress and decreased the ETS efficiency in platelet mitochondria. Although no changes were found in mitochondrial biogenesis, 6 week of HIT enhanced mitochondrial OXPHOS capacity and attenuated the HE-depressed mitochondrial ETS efficiency in platelets. Therefore, the HIT regimen effectively improve platelet bioenergetics, possibly by enhancing mitochondrial quality rather than quantity in platelets. Moreover, HIT also markedly reduces the enhancement of platelet-mediated TG by HE, which response is associated with alleviating the HE-induced mitochondrial dysfunction of platelets. These experimental findings may facilitate the identification of effective exercise training regimens to increase aerobic capacity and minimize platelet mitochondrial dysfunction and subsequent thrombus formation evoked by hypoxic stress.

Materials and Methods {#Sec14}
=====================

Subjects {#Sec15}
--------

The study was in accordance with the Declaration of Helsinki and approved by the Chang Gung Memorial Hospital Institutional Review Board, Taiwan. All experiments were performed in accordance with relevant guidelines and regulations. A total of 45 sedentary males who were non-smokers, did not use medications or vitamins, and were free of any cardiopulmonary/hematological risks were recruited from Chang Gung University, Taiwan. According to our previous studies, the menstrual phases of women influence activity of cardiac autonomic nervous system^[@CR26]^ and platelet reactivity^[@CR27]^. Hence, this study excluded female subjects to avoid the effects of gender and the menstrual phase on cardiovascular^[@CR26]^ and hematological^[@CR27]^ functions. No subjects had engaged in any regular physical activity (exercise frequency ≤ once weekly, duration \< 20 min) or had been exposed to high altitudes (≥altitude of 3000 m) for at least 1 year before the experiment. All subjects provided informed consent after the experimental procedures were explained. These subjects were randomly divided into three groups: the HIT (n = 15), MCT (n = 15), and CTL (n = 15) groups. Moreover, all subjects arrived at the testing center at 9:00 AM to eliminate any possible circadian effects. The experimental environment was maintained at a temperature of 22+/− 0.5 °C with a relative humidity of 60+/− 5%. Participants were instructed to fast for at least 8 hours and to refrain from strenuous physical exercise for at least 48 hours before sampling.

Training protocols {#Sec16}
------------------

Both the HIT and MCT groups performed exercise regimens on a stationary bicycle ergometer 5 times a week for 6 weeks. For comparison, CTL participants did not any undergo any exercise but were carefully monitored, and we recorded information on their physical activity and nutritional intake for 6 weeks. HIT subjects warmed up for 3 min at 30% of VO~2max~ before starting five exercise cycles, each lasting 3 min at 80% of VO~2max~ interspersed with a 3 min active recovery period at 40% of VO~2max~. The exercise session was terminated with a 3 min cool-down period at 30% of VO~2max~. The MCT group had the same warm-up and cool-down protocols as the HIT group, except that the training period was 30 min at 60% of VO~2max~ ^[@CR13],\ [@CR28]^. The two exercise protocols were isovolumic with the same exercise duration \[i.e., HIT exercise volume: 6 min (40%VO~2max~ + 80%VO~2max~) × 5 cycles = MCT exercise volume: 30 min (60%VO~2max~)\]. Each subject used a heart rate (HR) monitor (Tango, SunTech Medical) to obtain the assigned intensity of exercise. The work-rate of the bicycle ergometer was adjusted continuously to ensure that the intensity of exercise matched the target HR throughout the training period. The percentage of HR reserve (%HRR) is widely considered to be equivalent to the percentage of VO~2~ reverse for exercise prescription purposes^[@CR29]^. Accordingly, the target HRs of HIT and MCT were calculated using the following equations^[@CR29]^:$$\documentclass[12pt]{minimal}
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                \begin{document}$$\begin{array}{c} \% {\rm{HHR}}= \% ({\rm{peak}}\,{\rm{HR}}-{\rm{resting}}\,{\rm{HR}})+{\rm{resting}}\,{\rm{HR}}\end{array}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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                \begin{document}$${\rm{Target}}\,{\rm{HR}}\,{\rm{of}}\,{\rm{HIT}}=3-{\rm{minute}}\,{\rm{intervals}}\,{\rm{at}}\,40 \% \,{\rm{HHR}}\,{\rm{and}}\,80 \% \,{\rm{HRR}}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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                \begin{document}$${\rm{Target}}\,{\rm{HR}}\,{\rm{of}}\,{\rm{MCT}}={\rm{sustained}}\,60 \% \,{\rm{HRR}}$$\end{document}$$

All subjects recorded their daily activities and nutritional intake throughout the experiment using the International Physical Activity Questionnaire Short Form^[@CR30]^ and the Written Diet Record^[@CR31]^, respectively. The participants were instructed to refrain from extra regular exercise until the end of the study. Moreover, the participant compliance rates for the three interventions were 100%. All subjects completed the exercise intervention and/or tests at the beginning of the present study and after 6 weeks in the three groups.

GXT {#Sec17}
---

Subjects performed a GXT on a bicycle ergometer (Corival 400, Lode) to assess their aerobic capacity 4 days before and 4 days after the 6 week interventions^[@CR18],\ [@CR28]^. The GXT was composed of 2 min of unloaded pedaling followed by a continuous increase in work-rate of 30 W per 3 min until exhaustion (*i*.*e*., VO~2max~). The V~E~, VO~2~, and VCO~2~ were measured breath by breath with a computer-based system (MasterScreen CPX, Cardinal-health Germany). The defined VO~2max~ was required to achieve the following 3 of 4 criteria: (i) the level of VO~2~ increased less than 2 mL/kg/min over at least 2 min; (ii) HR exceeded its predicted maximum; (iii) the respiratory exchange ratio exceeded 1.2, and (iv) the venous lactate concentration exceeded 8 mM, consistently with the guidelines of the American College of Sports Medicine for exercise testing^[@CR32]^. The value of ventilatory threshold was determined by applying the following ventilatory criteria; (i) the V~E~/VO~2~ ratio increased without a corresponding increase in the V~E~/VCO~2~ ratio; (ii) PETO~2~ increased without a decrease in the PETCO~2~, and (iii) a departure from linearity for V~E~ ^[@CR32]^.

The HE test and blood collection {#Sec18}
--------------------------------

Each subject performed the HE test on the 2^nd^ day before the intervention and on the 2^nd^ day after the intervention in an air-conditioned normobaric hypoxia chamber (Colorado Mountain Room, Boulder, CO) as described in our previous studies^[@CR18],\ [@CR28]^. The hypoxia chamber was maintained at a temperature of 22+/− 0.5 °C with a relative humidity of 60+/− 5%; a CO~2~ scrubber eliminated CO~2~ in the air (\<3,500 ppm). The HE test on the bicycle ergometer required 50 W of warm-up for 3 min, an increase in the work-rate to 100 W with continuous exercise for 30 min, and then recovery to 50 W of a cool-down period for 3 min. During the test, the O~2~ concentration was set to 12%, which corresponds to an altitude of 4,460 m^[@CR19],\ [@CR29]^. For safety reasons, the HE test was terminated immediately when the level of O~2~ saturation dropped to \<70% or the subject complained of discomfort. All subjects were free of acute mountain sickness symptoms during the experimental period.

At rest and immediately after the HE test, 40 ml blood samples were collected from an antecubital vein using a clean venipuncture (20 gauge needle). The first 2 ml of blood was discarded, and the remaining blood was used to measure hematological parameters. Blood cells were counted using a Sysmax SF-3000 cell counter (GMI, Inc., Ramsey, MN).

Platelet isolation {#Sec19}
------------------

Blood samples were collected in polypropylene tubes that contained sodium citrate (3.8 g/dl, 1--9 vol. blood). PRP was prepared by centrifugation at 300 *g* for 10 min at room temperature (20 °C). Platelets were pelleted by centrifugation of the PRP at 1,500 *g* for 10 min and then washed once with phosphate buffered saline containing ethylenediaminetetraacetic acid (final concentration, 4 mM) (Sigma) to prohibit platelet activation. The number of platelets was adjusted to 2 × 10^8^ cells/ml with RPMI medium (Sigma). The tests of blood samples were repeated to ensure reproducibility. The analysis of platelet functions was completed within 2 hours after cell purification.

Mitochondrial content, MP, and MOB in platelets {#Sec20}
-----------------------------------------------

The relative quantification of platelet mitochondrial-localized dyes using the single-color flow cytometric analysis allows for the sensitive measurement of a variety of mitochondrial parameters, including mitochondrial content, MP, and MOB, as described in previous studies^[@CR18],\ [@CR33]^. The platelet suspensions (2 × 10^8^ cells/ml) were incubated with MitoTracker Green FM (a green-fluorescent mitochondrial stain, 200 nM) (Invitrogen), tetramethylrhodamine ethyl ester (TMRE, a mitochondrial potential-sensitive probe, 20 nM) (Invitrogen), or MitoSOX Red (a mitochondrial superoxide indicator, 6.6 μM) (Invitrogen) in the dark for 30 min at 4 °C. Then, the platelets were gated separately from other particles on the basis of forward/sideways scatter, and the mean fluorescence intensity from 100,000 events representing the platelets was calculated using a FACScan flow cytometer (Becton Dickinson).

Mitochondrial respiration and BHI in intact platelets {#Sec21}
-----------------------------------------------------

The mitochondrial O~2~ consumption of platelets (2 × 10^8^ cells/ml) in RPMI 1460 medium was measured using high-resolution respirometry (Oroboros O2K). Mitochondrial respiration coupled towards ATP production (ATP-linked OCR) was measured by the fall in O~2~ consumption following the addition of oligomycin (0.2 μg/ml), an inhibitor of ATP synthase. The remaining rate of mitochondrial respiration represents a proton leak that uncouples OXPHOS from the ETS. The total O~2~ consumption of platelets was measured at baseline and after the addition of the uncoupling agent carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 2 μM) to induce maximal O~2~ consumption. The difference between the basal and maximal respiration is called the reserve capacity of OCR. Non-mitochondrial respiration (non-mito OCR) was quantified by inhibiting mitochondrial respiration through the addition of rotenone (a mitochondrial Complex I inhibitor, 1 μM) and antimycin A (a mitochondrial Complex III inhibitor, 1 μM)^[@CR18],\ [@CR34]^.

The BHI was calculating from the result of coupling control protocol to quantify platelet mitochondrial function using the following equations^[@CR35]^:$$\documentclass[12pt]{minimal}
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                \begin{document}$$\mathrm{Log}[(\mathrm{ATP}-\mathrm{linked}\,{\rm{OCR}})\times ({\rm{Reserved}}\,{\rm{capacity}}\,{\rm{of}}\,{\rm{OCR}})/({\rm{Proton}}\,{\rm{leak}})\times (\mathrm{Non}-\mathrm{mito}\,{\rm{OCR}})]$$\end{document}$$ $$\documentclass[12pt]{minimal}
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                \begin{document}$$\mathrm{ATP}-\mathrm{linked}\,{\rm{OCR}}={\rm{Routine}}\,{\rm{state}}-{\rm{Leak}}\,{\rm{state}}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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                \begin{document}$${\rm{Reserved}}\,{\rm{capacity}}={\rm{ETS}}\,{\rm{state}}-{\rm{Routine}}\,{\rm{state}}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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                \begin{document}$$\mathrm{Non}-\mathrm{mito}\,{\rm{OCR}}={\rm{Residual}}\,{{\rm{O}}}_{2}{\rm{consumption}}\,({\rm{ROX}}){\rm{state}}$$\end{document}$$ $$\documentclass[12pt]{minimal}
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                \begin{document}$${\rm{Proton}}\,{\rm{leak}}={\rm{Leak}}\,{\rm{state}}-{\rm{ROX}}\,{\rm{state}}$$\end{document}$$

Mitochondrial respiration in permeablized platelets {#Sec22}
---------------------------------------------------

A substrate, uncoupler, inhibitor titration (SUIT) protocol was applied to permeablized platelets preparation and analyzed the respiration capacity of platelet mitochondria. By multiple substrate titration, electron flow from FAO and mitochondrial Complex I and II were well controlled, and the mitochondrial OCR of each state was measured by a high-resolution respirometry (Oroboros O2K)^[@CR18],\ [@CR34]^.

Intact platelets (2 × 10^8^ cells/ml) were incubate at 37 °C in the O2K chamber of mitochondria respiratory medium MiR05 (EGTA 0.5 mM, MgCl~2~ · 6H~2~O 3 mM, lactobionic acid 60 mM, taurine 20 mM, KH~2~PO~4~ 10 mM, HEPES 20 mM, D-sucrose 110 mM, and bovine serum albumin 1 g/l, pH = 7.1). Data acquisition was started after the oxygen flux stabilized. The O~2~ consumption in this state was the routine respiration from endogenous substrates in cells. Then the plasma membrane was permeabilized by digitonin (0.1 mg) titration after a concomitant addition of malate (2 mM) and palmitoyl-DL-carnitine-HCl (20 μM). Since the absence of adenylate in the chamber, the respiration in this state was caused by mitochondrial proton leakage (LEAK). The O~2~ consumption by FAO was evaluated by addition of 1 mM ADP (Calbiochem). Oxidative phosphorylation capacity of mitochondrial Complex I and II were acquired through the addition of NADH resources pyruvate (5 mM) and glutamate (10 mM) and SDH resource succinate (10 mM). A cytochrome c (10 μM) test was applied to evaluate whether the outer mitochondrial membrane was intact. The maximal convergent capacity of the ETS was subsequently obtained by FCCP titration (0.5 μM/steps).

Finally, the inhibitors for Complex I, II and III (0.1 μM rotenone, 5 mM malonic acid, and 0.5 μM myxothiazol/2.5 μM antimycin A) were progressively added to suppress the O~2~ consumption of platelets mitochondria. At last, tetramethyl-p-phenylenediamine (TMPD, 0.5 mM) applied as an artificial substrate for reducing cytochrome c, was added for cytochrome c oxidase activity measurement and then blocked by mitochondrial Complex IV inhibitor sodium azide (200 mM). All chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA) if not stated otherwise.

Enzyme activities of glycolysis and the Krebs cycle in platelets {#Sec23}
----------------------------------------------------------------

The activities of HK (Sigma), PDH (BioVision), and LDH (Sigma) in glycolysis and the activities of CS (BioVision) and SDH (BioVision) in the Krebs cycle of platelets (1 × 10^8^ cells/ml) were measured with commercially available colorimetric kits according to the manufacturer's instructions^[@CR18]^.

Dynamic TG assay {#Sec24}
----------------

The dynamic TG in PRP was measured by calibrated, automatic thrombinography (Synapse/Thrombinoscope BV, Maastricht, the Netherlands), as described in our earlier works^[@CR4],\ [@CR13]^. Oligomycin (2 μg/ml), FCCP (3.75 μM), or rotenone (0.1 μM) plus antimycin A, (2.5 μM) were added to the PRP (2 × 10^8^ cells/ml), which was then warmed to 37 °C for 10 min. Following incubation, eighty microliters of the PRP samples were allocated into the wells of round bottom 96-well microtiter plates (Nunc). Twenty microliters of reagent containing tissue factor (TF) was mixed with the plasma samples to obtain a final concentration of 0.5 pM TF. Coagulation was started by adding 0.1 M CaCl~2~ (20 μL) in a fresh mixture of fluobuffer (containing 20 mM HEPES and 60 mg/mL bovine serum albumin in pH 7.35) containing 2.5 mM Z-Gly-Gly-Arg-AMC (the fluorogenic substrate) (Synapse/Thrombinoscope BV, Maastricht, the Netherlands). Upon cleaving by thrombin, the fluorescent AMC (7-amino-4-methylcoumarin) is released and measured with a 390-nm-excitation and a 460-nm-emission filter set in an Ascent Fluoroskan (Thermo Fisher Scientific Inc., the Netherlands). All reagents were warmed to 37 °C before the experiment began. Fluorescence was recorded for 60 min. The fluorescence signal was corrected for substrate consumption, plasma colour variability and inner filter fluorescence effect by running in parallel calibrating wells where 80 microliters plasma samples were mixed with 20 microliters Thrombin Calibrator from Thrombinoscope BV.

The calculated data using analytic software from Synapse/Thrombinoscope BV were plotted and expressed in terms of lag time (time until initial thrombin formation, min), endogenous thrombin potential (ETP, area under the thrombin curve, nM × min), the peak height of thrombin (nM), and the rate of TG (mean slope = peak height/\[time to peak-lag time\], nM/min)^[@CR4],\ [@CR13]^.

Statistical analysis {#Sec25}
--------------------

The results are expressed as the mean ± SEM. The statistical software package StatView was used for data analysis. Experimental results in each group were analyzed by the repeated measures ANOVA and Bonferroni's post-hoc test to compare the count, mitochondrial OXPHOS, oxidative stress and biogenesis of platelets, as well as, the dynamic TG in PRP before and immediately after HE at the beginning of the present study and after 6 weeks. In addition, the comparison of cardiopulmonary fitness during GXT at the beginning of the present study and 6 weeks later in various groups was analyzed by the repeated measures ANOVA and Bonferroni's post-hoc test. The criterion for statistical significance was *P* \< 0.05.

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The authors would like to thank the volunteers for their enthusiastic participation in the present study. This work was supported by the National Science Council of Taiwan (grant number NSC 105-2314-B-182-013-MY3, 103-2314-B-182-005-MY3), Chang Gung Medical Research Program (grant number CMRPD3D0133, CMRPD1E0262), and Healthy Aging Research Center, Chang Gung University (grant number EMRPD1A0841).

Jong-Shyan Wang was involved in conception and design of research; Li-Hua Wu and Shao-Chiang Chang performed experiments; Jong-Shyan Wang and Li-Hua Wu analyzed data, interpreted results of experiments, prepared the Figures, and drafted the paper; Jong-Shyan Wang, Tieh-Cheng Fu, and Ching-Hui Huang edited and revised the paper; Li-Hua Wu, Shao-Chiang Chang, Tieh-Cheng Fu, Ching-Hui Huang, and Jong-Shyan Wang approved the final version of paper.

Competing Interests {#FPar1}
===================

The authors declare that they have no competing interests.


Alpha 3 is now available for download! The full change log is at the end of this post.

Devlog

Devlog Update 104! I guess that means 2 years of weekly devlogs, wow :)

Garret modeled a Motion Simulator ride during this weeks Art Stream. At just 2x2 tiles it is the smallest ride in the game and should work nicely to fill some gaps in parks that are running out of space.

Getting the animation working in the game with the hydraulics and all turned out to be a bit more difficult than originally expected, but here it is:

(Animation shortened for GIF)

And we added cloud shadows:

They’re fairly subtle but help visualize the current weather a bit better.

Team

I’ve been the sole programmer working on Parkitect so far and it would’ve been cool to finish it alone… there’s a bunch of work left though and our sale numbers are looking good enough that we can afford to bring on some people to lower the work load and speed things up a bit, so Luuk and Tim from ParkitectNexus are joining us part time to help with programming tasks.

Welcome to the team! :D

Alpha 3 Changelog

- added Motion Simulator

- added Shark costume for Entertainer

- added brick walls set and some industrial props

- added sounds for Giga Coaster, Mini Coaster, Alpine Coaster, Suspended Coaster, Monorail, Ghost Mansion Ride, Inverted Dark Ride

- added new path style

- added paths don’t connect through walls and fences anymore

- added paths don’t spawn handrails if wall/fence already exists in same location

- added working queue signs

- added text signs that can be put on paths and act as “no entry” signs

- added underwater tunnels

- added track tunnels automatically updating when terraforming

- added search bar for blueprints

- added embedding log flume tracks into water/terrain without spawning tunnels

- added stats graphs for tracked rides

- added cloud shadows

- added Dutch names

- improved the look of flowers

- improved the look of tunnels (look darker on the inside)

- improved savegame load times by up to ~50%

- fixed a case where guests could queue for a closed attraction

- fixed various issues with building fences

- fixed guests rarely thought an empty queue is full and didn’t enter it

- should fix very rare case where mechanics could get stuck in ride exit gates


In Egypt, football isn't merely a sport. It's a rite of passage for many of the country's youth and a national hobby that brings together Egyptians in a common goal regardless of status, class, social background and profession. When football season rolls around, you'll find most cafes, bars and lounges packed with the football fanatics of Egypt in support of their national team and as of late, in worship of our national treasure Mo Salah. Logically, then, the football field is the equal opportunity territory uniting Egyptian athletes based solely on merit and talent - or is it? Hany Ramzy (pictured above) is thought of as one of Egypt's greatest defenders and captained the Egyptian national team during a prolific career. But he's also the only Copt to do so.

Coptic Egyptian football player, Mena El Bendary, is a prime example - a young man that has suffered at the hands of the interfaith dynamics of Egyptian football. Insisting that he's faced religious prejudice - along with a number of other Copts - Bendary decided to take matters into his own hands and to tackle the problem at the root. Starting his own football academy in Alexandria -Je Suis - to create an opportunity for those who claim to have been put in a corner by coaches within official football clubs in Egypt, Bendary is adamant on making a change.

"I used to play at one of Egypt's main football clubs and I was recommended to them by a Muslim coach. A month in, the coach asked me to change my name so I left," says Bendary. "I tried and got rejected by a few other places before I decided to start my own academy."

With over 300 players in his academy, 22-year old Benadary is currently the youngest qualified coach in Egypt's history. He decided to sacrifice the pursuit of his own dreams in order to give younger generations a shot at reaching their goal. Bendary chose the name because it symbolises potential." It means I am. I chose it because I want these kids to be able to say I am capable, I am going to reach my potential and I am going to reachto my goal."

Why have there only been five Coptic players in the top levels of Egyptian football, if there's no discrimination?

Retired Egyptian football player, Ahmed 'Mido' Hossam is one of the people who have spoken about the matter publicly. In an interview with DMC Sport last April, the nomadic footballer asserted there has been an issue with the participation, or lack thereof, of Copts at Egyptian clubs. Speaking against the prejudice, Mido goes on to prove his point by inquiring as to "why have there only been 5 Copt players in the top levels of Egyptian football, if there's no discrimination?." Mido clarifies that a lot of these kids stop playing football as a result of the injustices they face by some coaches. Bendary hopes to save Copt children with footballing ambitions from the kind of rejection that ended his own dreams by creating an environment that fosters acceptance, opportunity and equality. The young coach has been improving on the academy which has been up and running for 3 years now and is working towards turning it into an official club.

"Je suis was initially founded for the Copts who'd gotten rejected because of their religion. Now we have both Copts and Muslims. We want to breed an environment of acceptance. We're all one community, Muslims and Christians. There's no difference when we play, it's only from some of the coaches in the clubs,” claims Bendary. "I need to make a change internally first because this isn't a problem limited to our generation. It's been ongoing for years. How many Copts do we have on our national teams? Barely any. I’d rather sacrifice my own career so that I can actually tackle the problem at the core rather than just have something fleeting."

The coach asked for my name and when I told him Mena, he said, maymesheesh ma3ak mostafa?

Je Suis doesn’t make profit as all the money goes towards expenses. Being an academy and not an official club, the fees for playing matches in the league are between EGP 1000 to 7000. Bendary started the organisation when he found that his was not an isolated case in Egyptian football, asserting that other Christian footballers have gone through variations of the young coach's rejection scenario.

“Honestly, it's frustrating and I don't get why this is the case here. We just want to play football,” says Bendary’s student, Mina Samir. “One of the many incidents that experienced was at a big club. The coach asked for my name and when I told him Mina, he said mayemsheesh ma3ak Mostafa (Does Mostafa work for you)?”

While Samir persisted in going to try-outs despite several dismissals, there are cases of those who simply gave up.

"Je Suis gives us hope. I was lucky to find the academy and I'd never give up football anyway. Even after I got rejected, I continued playing in church tournaments and in Alexandria's football academy. With my brother, it was different though. The first few times he got rejected because of his faith, he decided to quit and change sports completely. Now he's a weightlifter," says Samir.

When he found out my name, he told me he’d call me to let me know if I made it and give me the details. He didn’t even take my phone number

What magnifies the issue even more for Bendary and Samir is that the issue doesn't seem to exist within their social circles.

"A lot of my friends are Muslim and we play football together all the time. Every Saturday, we play a few matches together so I don’t know why some of the coaches differentiate when it comes to professional clubs. It's also not all Muslim coaches, to be fair. Besides, no matter what, I'm going to keep persisting. Look at Mohamed Salah and what he went through to get to where he is. He made it. And he's a Muslim playing among Christians. His religion never made a difference," concludes Samir.

Mena Ayman is another one of Bendary's students who's found solace in Je Suis.

“I passed the trials for the first round of tryouts at one of the clubs and was asked by the captain to sign the papers taking me to the second round. What usually happens once you’re asked to sign your name is that you’re given a paper with the details for the next round and all you have to do is show up. When he found out my name, he told me he’d call me to let me know if I'd made it and give me the details. He didn’t even take my phone number,” Ayman told us.

Irrespective of age, these Egyptians speak out on facing the same issue despite not giving up and persisting in going to try-outs. 26-year old Melad Gaber had been playing for an official club for two years before facing a similar scenario.

“I was playing with a club for 2 years before a new coach was hired. He asked me to change my name. I told him no and I stopped playing football,” explains Mena. “After that incident, I stopped going to try-outs for a while. That is, until I found Je Suis.”

It could be years before Bendary's efforts bear fruit, but what his ambitions offer kids like Mena is a light at the end of tunnel.

Main image taken from Scoopnest.com


const { expect } = require('chai');
const { expectRevert, BN, balance } = require('@openzeppelin/test-helpers');

const EtherToken = artifacts.require('EtherToken');

contract('EtherToken', accounts => {
    let token;
    const name = 'Ether Token';
    const symbol = 'ETH';
    const sender = accounts[0];
    const receiver = accounts[1];

    beforeEach(async () => {
        token = await EtherToken.new(name, symbol);
    });

    it('verifies the token name after construction', async () => {
        expect(await token.name.call()).to.eql(name);
    });

    it('verifies the token symbol after construction', async () => {
        expect(await token.symbol.call()).to.eql(symbol);
    });

    it('verifies the balance and supply after a deposit through the deposit function', async () => {
        const value = new BN(1000);
        await token.deposit({ value });

        const balance = await token.balanceOf.call(sender);
        expect(balance).to.be.bignumber.equal(value);

        const supply = await token.totalSupply.call();
        expect(supply).to.be.bignumber.equal(value);
    });

    it('verifies the balance and supply after a deposit through the fallback function', async () => {
        const value = new BN(122);
        await token.send(value);

        const balance = await token.balanceOf.call(sender);
        expect(balance).to.be.bignumber.equal(value);

        const supply = await token.totalSupply.call();
        expect(supply).to.be.bignumber.equal(value);
    });

    it('verifies the balance and supply after a deposit through the depositTo function', async () => {
        const value = new BN(4);
        await token.depositTo(receiver, { value });

        const balance = await token.balanceOf.call(receiver);
        expect(balance).to.be.bignumber.equal(value);

        const supply = await token.totalSupply.call();
        expect(supply).to.be.bignumber.equal(value);
    });

    it('verifies the balance and supply after a withdrawal', async () => {
        const value = new BN(100);
        await token.deposit({ value });

        const value2 = new BN(20);
        await token.withdraw(value2);

        const balance = await token.balanceOf.call(sender);
        expect(balance).to.be.bignumber.equal(value.sub(value2));

        const supply = await token.totalSupply.call();
        expect(supply).to.be.bignumber.equal(value.sub(value2));
    });

    it('verifies the ether balance after a withdrawal', async () => {
        const value = new BN(500);
        await token.deposit({ value });

        const prevBalance = await balance.current(sender);

        const value2 = new BN(200);
        const res = await token.withdraw(value2);
        const transaction = await web3.eth.getTransaction(res.tx);

        const newBalance = await balance.current(sender);
        const transactionCost = new BN(transaction.gasPrice).mul(new BN(res.receipt.cumulativeGasUsed));
        expect(newBalance).to.be.bignumber.equal(prevBalance.sub(transactionCost).add(value2));
    });

    it('verifies the ether balance after a withdrawal to target account', async () => {
        const value = new BN(800);
        await token.deposit({ value });

        const prevBalance = await balance.current(receiver);

        const value2 = new BN(56);
        await token.withdrawTo(receiver, value2);

        const newBalance = await balance.current(receiver);
        expect(newBalance).to.be.bignumber.equal(prevBalance.add(value2));
    });

    it('verifies the balances after a transfer', async () => {
        const value = new BN(300);
        await token.deposit({ value });

        const value2 = new BN(10);
        await token.transfer(receiver, value2);

        const senderBalance = await token.balanceOf.call(sender);
        expect(senderBalance).to.be.bignumber.equal(value.sub(value2));

        const receiverBalance = await token.balanceOf.call(receiver);
        expect(receiverBalance).to.be.bignumber.equal(value2);

        const supply = await token.totalSupply.call();
        expect(supply).to.be.bignumber.equal(value);
    });

    it('should revert when attempting to transfer to the token address', async () => {
        const value = new BN(500);
        await token.deposit({ value });

        await expectRevert(token.transfer(token.address, new BN(1)), 'ERR_ADDRESS_IS_SELF');
    });

    it('should revert when attempting to transferFrom to the token address', async () => {
        const value = new BN(111);
        await token.deposit({ value: value });

        const value2 = new BN(10);
        await token.approve(receiver, value2);

        await expectRevert(token.transferFrom(sender, token.address, new BN(1), { from: receiver }),
            'ERR_ADDRESS_IS_SELF');
    });
});


Landmark Kirwan commission delays plan on school funding

A plan to revamp and increase spending on Maryland public schools will be delayed beyond next year’s General Assembly session, officials said Wednesday.

Lawmakers were set to spend the first few months of 2018 debating the first major overhaul of education policy in more than 15 years. Instead, that discussion will be pushed back until after the 2018 state elections.

William E. “Brit” Kirwan, leader of the commission issuing the policy recommendations, said Wednesday that his panel is moving toward consensus on recommending universal pre-K, a revamped pay structure for teachers and a new formula to more fairly distribute education funding across the state, among other measures.

However, there’s not enough time to calculate the costs of the commission’s recommendations or come up with suggestions for paying for them before the new legislative session begins in January, Kirwan said.

“There’s simply no point in producing a report the state can’t afford,” he said. “The alternative [to delaying it] is to have a wonderful report that is not based in fiscal reality.”

Sean Johnson, political director for the state’s largest teachers union, called the delay disappointing because it means unmet needs in schools will linger for at least another year. But he said it was more important to present changes that lawmakers feel comfortable approving.

“This is a once-in-a-generation opportunity,” Johnson said. “And if it takes a few more months to get it right, it’s worth it.”

The legislature created the Commission on Innovation and Excellence in 2016 to explore two questions: Should the state revise the 2002 funding formula that distributes school aid? And what major new education policies should the state enact to put Maryland public schools on a par with the best in the world?

The commission had a December 2017 deadline to answer those questions, and lawmakers put Kirwan, former chancellor of the University of Maryland system, in charge of the process.

The commission became known as the Kirwan Commission, and its recommendations have been highly anticipated in education circles. Nearly all of the eight Democrats running for governor have cited the impending report as they’ve discussed how to improve education in the state.

CAPTION

A ceremony is held for students graduating from high school through the Re-engagement Center. (Kenneth K. Lam, Baltimore Sun video)

A ceremony is held for students graduating from high school through the Re-engagement Center. (Kenneth K. Lam, Baltimore Sun video)

CAPTION

Maryland schools superintendent has rejected Verletta White for permanent Baltimore County school superintendent due to ethics violations as well as the school system’s failure to complete an audit of how it awards millions of dollars in contracts. (Algerina Perna, Baltimore Sun video)

Maryland schools superintendent has rejected Verletta White for permanent Baltimore County school superintendent due to ethics violations as well as the school system’s failure to complete an audit of how it awards millions of dollars in contracts. (Algerina Perna, Baltimore Sun video)

A consultant hired by the commission to review the current funding formula, known as the Thornton formula, determined that Maryland would need to spend about $2.9 billion more each year on K-12 education if all students were to have equal access to high-quality schooling. About $2 billion of that amount would need to come from the state, consultants said. About $7.9 billion of of the state’s roughly $43 billion annual operating budget is spent on K-12 schools.

That price tag does not take into account changes the Kirwan Commission would recommend to improve schools — proposals such as creating a way for quality teachers to earn more without having to leave their classrooms for higher-paying jobs in school administration, providing free or low-cost pre-kindergarten to every 4-year-old child in the state, giving more resources to at-risk students, and reinstating vocational training in many high schools.

Kirwan declined to say Wednesday whether the changes favored by the commission would have a price tag in the hundreds of millions or billions of dollars.

Increasing education funding by hundreds of millions or billions would likely require a funding stream to pay for it, according to budget analysts.

Delaying the report until the second quarter of 2018 means there will not be enough time in the coming General Assembly session to enact the changes before it is scheduled to adjourn in April.

It also means that state lawmakers and Gov. Larry Hogan would not have to vote on a potentially controversial school funding measure immediately ahead of an election year.

The Kirwan Commission does not expire until June 2018.

Kirwan said that a complex, and potentially costly, overhaul of education will require a lot of “persistence” to enact

“A silver lining in taking a little more time is it gives us a chance to make the work of the commission known throughout the state,” Kirwan said.


This item is in very nice condition. Made of solid oak construction and very sturdy. Has a center drawer made of solid wood and dovetail construction. The legs are Queen Anne styling. There are some minor scratches seen on the piece however barely noticeable. The piece stands approx 17 3/4" high with a top measurement of 45" x 24". The wood grain is fantastic and the coloration is a natural to medium oak finish without a high gloss.
Shipping is available on this item please provide your zip code when asking for a quote.. See more Coffee Tables for Sale in Willimantic

This item is in very nice condition. Made of solid oak construction and very sturdy. Has a center drawer made of solid wood and dovetail ...


Severe violence by radical protesters

Police exercise restraint in law enforcement

Freedom and rule of law

Reject foreign interference into HKSAR's affairs

The Hong Kong Special Administrative Region (HKSAR) Government today (January 1) refutes as unfounded and misguided claims in an open letter to the Chief Executive signed by a group of overseas politicians and persons.A Government spokesman made clear that since Hong Kong's return to the Motherland, "One Country, Two Systems" and a high degree of autonomy had been faithfully implemented in strict accordance with the Basic Law of HKSAR. The Basic Law clearly stipulates that the HKSAR is an inalienable part of the People's Republic of China (PRC). Foreign politicians and organisations should not interfere in any form in the internal affairs of the HKSAR."We are gravely concerned that the claims in their letter are biased and misleading. The HKSAR Government must rebut them to ensure they truly understand the extent of violence by radical protesters and the attacks they have made on the Police and citizens," the spokesman said.Over the past six months, it was widely reported (though less so in overseas media) that protester violence had escalated and reached an alarming level. Masked rioters have vandalised public facilities and shops, committed arson, hurled petrol bombs as well as wantonly attacked police officers and people with different views in various districts, seriously undermining public order. An innocent man was set on fire in broad daylight by protesters while a government contract cleaner was killed by a brick thrown at him. So far, 520 police officers have been injured on duty.No protesters have been killed as a result of direct Police action, while several policemen have been seriously injured. This is the stark reality that foreign politicians and organisations, including the signatories of the open letter, must be made aware of.The extent of students and minors being involved in such violent protests is particularly worrying. Two secondary school students have been arrested for bringing a highly explosive substance, triacetone triperoxide (TATP), to their school in Ma On Shan. The explosive substance was safely removed by Police.While TATP may look like an innocuous white powder, it is very destructive and unstable. This case shows that dangerous items and explosives have fallen into the hands of young students.And just days ago, a 16-year-old teenager was arrested for assaulting police officers and robbery for attempting to snatch a gun from a police officer. He has been refused bail by the court.The severity of the situation has often brought our city to a chaotic halt, with many citizens suffering from severe disruption to daily life and fear for their personal safety. This fear is not confined to physical violence, but to the immense pressure of being bullied or harassed simply because of their background and opinions.The spokesman stressed that Police do not initiate actions against protesters and only respond with appropriate and proportionate force when protesters take part in illegal activities.The actions and response of Police over the Christmas period, and at all times over the past six months, were only in response to the blatant disregard for public safety and order by radical protesters. Police acted with restraint and the minimum force necessary to restore law and order. Police have established clear guidelines on the use of force which are consistent with international human rights norms and standards.Since June 9, over 1 000 demonstrations, processions and rallies have been held in Hong Kong. All along, Police have facilitated the conduct of peaceful and lawful public meetings or public processions. However, if protesters resort to violence, the Police have no choice but, if there are no other means and after giving warnings where circumstances permit, to use appropriate force to prevent injury to life and property.If Police are unable to carry out any operations to arrest rioters resorting to escalating violence, causing damage from one place to another or even manufacturing life-threatening weapons, we will not be able to competently deal with the difficult and unprecedented situation confronting our city.The spokesman stressed that, "Hong Kong is fully capable of resolving the current challenges on its own and has a well-established mechanism to deal with complaints against police officers through the Independent Police Complaints Council"."As a matter of principle and law, the HKSAR Government rejects and deplores the use of violence against anyone for any purpose and on any occasion. Violence is after all violence. We have the responsibility to adopt lawful measures to maintain public safety, public order, as well as safeguard people's life and property. It is necessary for us to actively investigate illegal and violent acts to bring offenders to justice, whoever they may be."The spokesman cited a recent judgement handed down on November 15 by Hong Kong's Court of First Instance which observed that criminal activity would not cease to be criminal activity simply because the actor believed himself or herself to be acting for a cause.The spokesman further stated, "The HKSAR Government fully recognises that freedom and the rule of law are the core values of Hong Kong and the cornerstone of our long-term prosperity and stability, and the HKSAR Government is determined to safeguard these core values at all times."In fact, the extensive media coverage – local, international and in the digital world – about the protests and events is unprecedented in Hong Kong's history. That in itself is evidence enough that the freedom of the press is well protected, and citizens of Hong Kong continue to enjoy the freedom of participating in lawful and peaceful demonstrations to express their views."In Hong Kong, we believe in the rule of law which means people are innocent until proven guilty; that offenders are only charged if there is enough evidence to do so; and we place our faith and trust in the integrity of our fiercely independent judiciary," the spokesman said, noting that Hong Kong's judicial independence ranked second in Asia and eighth in the world, according to the World Economic Forum: Global Competitiveness Report."Those who claim that people have been unjustly detained are simply jumping the gun and conveniently ignoring the tried and trusted mechanisms that underpin our common law system."Nobody anywhere is in any position to claim that protesters have been unjustly detained until such time as the legal process has run its course."The spokesman rejected outright the suggestion in the open letter that the international community establish an international independent inquiry mechanism."Such action, coupled with the threat of Magnitsky sanctions, would be a gross interference in Hong Kong's governance and autonomy as well as the sovereignty of our nation. No government would tolerate such action or bow to such pressure," the spokesman added.On political reform, the spokesman said this was also an internal matter for the HKSAR Government."We did introduce legislation to make the selection of the 2017 Chief Executive election more democratic only to have it rejected by those politicians claiming to be championing for democracy," the spokesman said.The spokesman emphasised that, "the HKSAR Government will continue to engage different sectors of society through the usual channels, whilst broadening and rejuvenating its communication with the public."Since September last year, the Chief Executive and Principal Officials of the Government have started a dialogue with the public through various channels."The Government is also establishing an Independent Review Committee comprising experts and community leaders. The Committee will look into the causes and full circumstances of the social unrest and probe Hong Kong's deep-seated social conflicts, from affordable housing, land supply, wealth gap, upward social mobility and opportunities for our young people to social justice."The Committee will recommend ways to address the real and long-entrenched community concerns that underlie the discord."We understand that many in the international community are concerned about Hong Kong and its future but Hong Kong people have the wisdom and resilience to solve the current impasse on our own. And we will," the spokesman added."We also understand that Hong Kong is being used as a pawn by some in the West to further their own agendas based on a one-sided narrative that paints a negative picture of the HKSAR Government's actions to restore calm and peace while ignoring the ongoing serious threats to law and order by radicals that they would hardly tolerate in their own country."

Ends/Wednesday, January 1, 2020

Issued at HKT 1:02

NNNN


Panagiotis Retsos

Panagiotis Retsos (, born 9 August 1998) is a Greek professional footballer who plays as a defender for Premier League club Sheffield United, on loan from Bayer Leverkusen of the Bundesliga, and the Greece national team.

Club career
Retsos was born in Johannesburg to  Greek parents, Christos and Eleni. In the summer of 2016, at age 18, he was promoted from Olympiacos' youth team by first manager Víctor Sánchez, who was impressed by his stamina. On 25 August 2016 he made his debut with the club in a Europa League play offs home match against Arouca.

On 8 February 2017, Retsos has appointed captain of the club in a Greek Cup goalless home draw against Atromitos. Retsos became the youngest captain of a Greek champion club aged only 18, five months and 30 days.

On 30 August 2017, the 19-year-old Retsos was sold to Bayer Leverkusen for a fee of £15.75 million. The Bundesliga outfit believe they have signed themselves a future star, with the teenager having already broken records in his career. The 19-year-old centre-back has signed a five-year deal with the Bundesliga outfit after Leverkusen beat off competition from Ligue 1 sides Stade Rennes and Lyon for his signature. On 17 September 2017, he made his debut in Bundesliga in a 4–0 home win game against SC Freiburg as a starter. On the club's Twitter account, Retsos was voted the Man of the Match.

On 9 April 2018, substitute Retsos scored his first goal in Bundesliga by firing into the far corner after a left-wing free-kick broke to him at the far post, in a 4–1 away win against RB Leipzig. It was  his first goal in his professional career. In the Bundesliga this season, Retsos has made a total of 14 starts so far and has also made six substitute appearances. He has impressed during this time, displaying composure at the back for the German outfit.

On 2 August 2018, Retsos suffered a heavy blow to the quadriceps during pre-season training with Bayer Leverkusen and according to the initial medical evaluation, he was diagnosed with tendonitis in the right thigh. The injury is expected to sideline the defender for at least six weeks. It is Retsos’ first serious injury with the German club and he will miss the kick-off of the 2018–19 Bundesliga season. On 26 October 2018, Retsos is available for selection again and was on the Bayer Leverkusen bench in a UEFA Europa League match against FC Zurich.

On 29 November 2018, after a very difficult period for the defender marred by injury (208 days to be exact), Retsos returned to action in a UEFA Europa League home game against Ludogorets, but was unlucky as in the first half hour left the pitch with a muscular injury on his left thigh and according to the initial reports, Retsos will now spend another 10-12 weeks on the sidelines. On 29 October 2019, after a second very difficult period for the defender marred by injury (334 days to be exact), played in a DFB-Pokal game as a substitute in a 1–0 home win against Padeborn.

On 1 February 2020, Retsos signed for Premier League club Sheffield United on loan from Bayer Leverkusen until the end of the 2019–20 season.

International career
Retsos, being born in South Africa, was eligible to represent South Africa. However, he decided to represent Greece, as he and his family returned to their native Greece when Retsos was aged one.

He made his debut for Greece on 31 August 2017, in a 0–0 home draw against Estonia in 2018 FIFA World Cup qualification.

Career statistics

Honours
Olympiacos
Super League Greece: 2016–17

Individual
Super League Greece Best Young Player of the Year: 2016–17

References

External links

Category:1998 births
Category:Living people
Category:Sportspeople from Johannesburg
Category:Greek footballers
Category:Greece youth international footballers
Category:Greece under-21 international footballers
Category:Greece international footballers
Category:Association football defenders
Category:Olympiacos F.C. players
Category:Bayer 04 Leverkusen players
Category:Sheffield United F.C. players
Category:Superleague Greece players
Category:Bundesliga players
Category:Greek expatriate footballers
Category:Expatriate footballers in England
Category:Expatriate footballers in Germany
Category:Greek expatriate sportspeople in England
Category:Greek expatriate sportspeople in Germany


I No Longer Bow to the Dark Master/TDM primal journal

Hello, my name is cj and I'm a reformed chocoholic (my Dark Master ). I went primal about 5 months ago.

I spent my adulthood up to now believing fully that a low-fat, low-animal-products diet was the healthy diet we should all follow for optimal health. However, every time I would re-commit to a "healthy" diet, I would only last a few days (or for one memorable vegan phase around my 40th birthday, 6 months) before no longer being able to bear the intense hunger all those carbs stimulated in me, or at I would resume eating a whole lot of processed, sweetened junk in addition to my high grains/legumes/low fat diet.

This year at my physical, my fasting blood sugar was 106. I am a physician assistant, so I knew this was not normal (*despite no comment from my doc) and looking back it has been that high at for least 1 or two years. I became determined to change my life. I don't remember exactly how I came across PB but I decided instantly to try it, because what the hell...my repeated efforts at low fat dieting were NOT WORKING. I think I started eating 70-80% primally at the very next meal.

About a year ago, I read some helpful advice on zenhabits.net to start with small changes in order to create new habits. I have used this strategy to slowly change several habits:

1. I changed from M&M's to cherries or grapes, then to one piece of dark chocolate after lunch and dinner. I'm now to the point where I can go without any dark chocolate after meals.

2. I started exercising 15 minutes 6 days a week for my New Years' resolution this year. Now I usually exercise more like 30-60 minutes 6 days a week, sometimes up to 2.5 hours if I have time. (usually walking or mountain biking a nearby wooded trail).

After 3 months of following PB I lost 20+ pounds, and have had absolutely no difficulty sticking with the PB diet or with keeping off every pound I've lost. But the weight loss plateaued at the beginning of summer, and though my waist is very slowly continuing to whittle down, it is time to make some further changes to lose some more unwanted blubber.

My new 30-day goal is to stop snacking between meals. I'm getting close-I've cut out the late morning snack by delaying breakfast time, and I don't feel like snacking after a good dinner. But the time between lunch and dinner is my weak spot, andt the time I am most likely to eat something like a Clif Builder bar, which tastes good and is satisfying, but is so not primal.

My stomach gets hungry out of habit. It is used to being fed at 4pm. I love the simplicity of not having hunger meltdowns on PB, and now I am ready to taste the sweet success of being a non snacker, 100%.

I design websites and blogs for a living. If you would like a blog or website designed by someone who understands Primal, see my web page.

Primal Blueprint Explorer My blog for people who are not into the Grok thing. Since starting the blog, I have moved close to being Archevore instead of Primal. But Mark's Daily Apple is still the best source of information about living an ancestral lifestyle.

Day 3, mission accomplished. I am glad I started this journal,because the thought of posting a failure was the only thing stopping me from a snackfest today.

I had some pita bread yesterday and my nose is stuffed up today. I never would have believed there was a connection between flour and my chronic congestion, but I prove it every time I have a piece of bread (which is not often, but still.). It's so nice not to spend every morning blowing my nose and sneezing. It's just one of many health benefits of PB I have experienced.

Day 4, a little easier today. When I exercise in the afternoon on an empty stomach, I feel a little weak and sweaty (and this is just light hiking for 30-60 minutes). I wonder if it is all in my head? I was always convinced I "couldn't" exercise when I was hungry.

Day 5-6: camped, hiked, and biked my way through Acadia National Park this weekend with nary a snack. I had no idea it would be so easy. No more anxious thoughts of when I can eat again. It doesn't matter! I will survive with no uncomfortable sweaty shaky hunger attacks. I'm sure it's a function of plenty of fat and protein in my meals. I <3 PB.

I'm already brainstorming my next 30-day challenge. But first, to complete this one. The Snack Queen abdicates her throne!

Day 7-8: it was a bit more difficult today but I could not admit defeat in front of Darth Friendly, so I controlled my perceived hunger and went mountain biking to get away from the fridge. Once again I had none of the expected weakness or fatigue with exercising on an empty stomach. This PB life continues to blast away at my preconceived notions of what I can and cannot do.


Q:

Publicly available genome sequence database for viruses?

As a small introductory project, I want to compare gene sequences of  different strains of flu virus.
Are there publicly available databases of flu virus gene sequences?

A:

There area few different influenza virus database resources:

The Influenza Research Database (IRD) (a.k.a FluDB - based upon URL)

A NIAID Bioinformatics Resource Center or BRC which highly curates the data brought in and integrates it with numerous other relevant data types

The NCBI Influenza Virus Resource

A sub-project of the NCBI with data curated over and above the GenBank data that is part of the NCBI

The GISAID EpiFlu Database

A database of sequences from the Global Initiative on Sharing All Influenza Data. Has unique data from many countries but requires user agree to a data sharing policy.

The OpenFluDB

Former GISAID database that contains some sequence data that GenBank does not have.

For those who also may be interested in other virus databases, there are:

Virus Pathogen Resource (VIPR)

A companion portal to the IRD, which hosts curated and integrated data for most other NIAID A-C virus pathogens including (but not limited to) Ebola, Zika, Dengue, Enterovirus, and Hepatitis C

LANL HIV database

Los Alamos National Laboratory HIV database with HIV data and many useful tools for all virus bioinformatics

PaVE: Papilloma virus genome database (from quintik comment)

NIAID developed and maintained Papilloma virus bioinformatics portal

Disclaimer: I used to work for the IRD / VIPR and currently work for NIAID.

A:

In addition to what others have suggested, I would also recommend PaVE as a resource. This is a curated database maintained by the NIAID and current holds over 300 papilloma virus genomes.


1. Technical Field
The present invention relates to a rolamite sensor for sensing deceleration of a vehicle, and particularly to a sensor for actuating a vehicle occupant restraint such as an air bag.
2. Description of the Prior Art
A known rolamite sensor has a movable member, preferably a roller, which moves along a guide surface in response to deceleration of a vehicle in which the sensor is mounted. The sensor has two independent electrical circuits. Each circuit uses an S-shaped band which at least partially encircles the roller. Each band is welded at one end to a tensioner fixed to a base. The tensioners protrude upwardly from the base above the guide surface for the roller. The tensioners take up a significant amount of space and require the sensor to have a relatively large enclosure.
Another known rolamite sensor has two S-shaped bands which at least partially encircle the roller, but only one electrical circuit. The roller is electrically conductive and has a permanent magnet core. An electromagnet is operative to repel the roller. When the roller is repelled by the electromagnet, the circuit should be completed. Thus, the electromagnet is operated to test the sensor. The electromagnet has a coil wound around a plastic bobbin on a multi-piece magnetic core. The electromagnet is disposed beyond one end of the path of movement of the roller, and takes up a substantial part of the overall volume of the sensor. The electromagnet is fixed to the plastic base of the sensor, and the sensor is much larger than if the electromagnet were omitted.


Q:

How to resolve this error? - Class 'ViewController' has no initializers

I have created UILabel programmatically in swift but it gives me following error :

Class 'ViewController' has no initializers

Code :
class ViewController: UIViewController {

    let lbl_LastName: UILabel!

    override func viewDidLoad() {
        super.viewDidLoad()
        lbl_LastName.frame = CGRectMake(10, 230, 300, 21)
        self.view.addSubview(lbl_LastName)
    }
}

A:

change let lbl_LastName: UILabel! to var lbl_LastName: UILabel!