A cost-effective plan for global testing - an infection rate stratified, algorithm guided, multiple-level, continuously pooled testing strategy.

The most effective measure to prevent or stop the spread of infectious diseases is the early identification and isolation of infected individuals through comprehensive screening. At present, in the COVID-19 pandemic, such screening is often limited to isolated regions as determined by local governments. Screening of potentially infectious individuals should be conducted through coordinated national or global unified actions. Our current research focuses on using resources to conduct comprehensive national and regional regular testing with a risk rate based, algorithmic guided, multiple-level, pooled testing strategy. Here, combining methodologies with mathematical logistic models, we present an analytic procedure of an overall plan for coordinating state, national, or global testing. The proposed plan includes three parts 1) organization, resource allocation, and distribution; 2) screening based on different risk levels and business types; and 3) algorithm guided, multiple level, continuously screening the entire population in a region. This strategy will overcome the false positive and negative results in the polymerase chain reaction (PCR) test and missing samples during initial tests. Based on our proposed protocol, the population screening of 300,000,000 in the US can be done weekly with between 15,000,000 and 6,000,000 test kits. The strategy can be used for population screening for current COVID-19 and any future severe infectious disease when drugs or vaccines are not available.

Resting MAPK expression in chronically trained endurance runners.

PURPOSE: There is a paucity of research investigating the expression of mitogen-activated protein kinases (MAPK) in chronically trained (CT) athletes. Thus, it is unclear how MAPK may contribute to performance and muscle adaptation in CT subjects. The purpose of this study was to determine MAPK total protein, and phosphorylated expression of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38-MAPK (p38) between untrained, and chronically trained runners. METHODS: Tissue samples were analysed from sedentary (SED; n = 5) controls and chronically trained runners (CT; n = 5). Resting muscle biopsy samples were analysed for total-MAPK - and ratio of phosphorylated/total (p-MAPK) - ERK1/2, JNK, and p38-MAPK via western blotting. Mann-Whitney U tests and effect sizes were utilized to determine differences in total MAPK protein content and phosphorylation status between SED and CT subjects. RESULTS: There was no difference in total-MAPK expression between SED and CT (p > .05). p-p38-MAPK tended to be greater for CT compared to SED (p = .07). There were moderate effect sizes of decreased pERK/total-ERK (d = -0.69) and increased pJNK/total-JNK (d = 0.54) in CT compared to SED. There was a positive correlation between p-p38-MAPK/total-MAPK and the percentage of type I fibres (r = 0.73, p = .016). CONCLUSION: Contrary to previous studies, chronic endurance training does not greatly influence total MAPK protein expression in chronically trained runners. However, resting phosphorylation of p38-MAPK may contribute to enhanced oxidative metabolism at chronically trained levels. These alterations are likely involved in the different physiological adaptations that occur following long-term training or at highly competitive levels.

Changes in resting mitogen-activated protein kinases following resistance exercise overreaching and overtraining.

PURPOSE: Many physiological maladaptations persist after overreaching and overtraining resistance exercise (RE). However, no studies have investigated changes in mitogen-activated protein kinases (MAPK) after overtraining in humans, despite their critical role regulating exercise-induced muscular adaptations. The purpose of this study was to describe the changes in total and resting phosphorylation status of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38-MAPK following a period of RE overreaching or overtraining. METHODS: Following 2-4 weeks of normal training (low volume/low intensity), two groups of males performed either a high-power overreaching protocol (HPOR n = 6, mean ± SD, age 23 ± 3.4 years, mass 86.5 ± 17.7 kg, height 1.77 ± 0.06 m) or high-intensity overtraining protocol (HIOT n = 8, age 19.8 ± 1.8 years, mass 76.8 ± 6.7 kg, height 1.8 ± 0.06 m). Resting muscle biopsies were obtained at baseline (BL; end of normal training period) and 24 h after the final session of stressful training (i.e., HPOR or HIOT programs). Total MAPK and ratio of phosphorylated/total (p-MAPK)- ERK1/2, JNK, and p38-MAPK were analyzed via western blotting. 2 × 2 (group × time) ANOVA determined differences in MAPK between BL and post-training protocols. RESULTS: Compared to BL, total-ERK increased after HPOR, but decreased after HIOT (p ≤ 0.05). p-ERK1/2/total-ERK increased after HIOT (p ≤ 0.05). The ratio of p-JNK/total-JNK and p-ERK1/2/total-ERK decreased after HPOR (p ≤ 0.05); however, this result was primarily due to increased total MAPK content. p-p38-MAPK decreased after HPOR (p ≤ 0.05). CONCLUSION: Total and p-MAPK are differentially expressed after HPOR and HIOT RE. These changes are likely involved in the maladaptation reported in overreaching and overtraining exercise. This is the first study describing altered MAPK in RE overtrained and overreached humans.

Resting extracellular signal-regulated protein kinase 1/2 expression following a continuum of chronic resistance exercise training paradigms.

Extracellular signal-regulated protein kinase 1/2 (ERK1/2) moderates skeletal muscle growth; however, chronic responses of this protein to unique resistance exercise (RE) paradigms are yet to be explored. The purpose of this investigation was to describe the long-term response of ERK1/2 following circuit weight training (CWT), recreationally weight training (WT), powerlifting (PL) and weightlifting (WL). Independent t-tests were used to determine differences in trained groups compared to sedentary controls. Total ERK1/2 content was lower in PL and WL compared to their controls (p ≤ 0.05). Specific trained groups displayed large (WL: pERK/total-ERK; d = 1.25) and moderate (CWT: total ERK1/2; d = 0.54) effect sizes for altered kinase expression compared to controls. The results indicate ERK1/2 expression is down-regulated after chronic RE in well-trained weightlifters and powerlifters. Lower expression of this protein may be a method in which anabolism is tightly regulated after many years of high-intensity RE.

High-power resistance exercise induces MAPK phosphorylation in weightlifting trained men.

Power is critical to muscle performance, specifically in athletic populations. Mitogen-activated protein kinase (MAPK) pathways (extracellular signal-regulated protein kinase (ERK 1/2), p38, and c-Jun NH(2)-terminal kinase (JNK)) are intracellular signal transduction mechanisms that partially regulate exercise-induced skeletal muscle alterations. These pathways are highly responsive to exercise, but their reaction to high power, multi-joint resistance exercise is yet to be examined. Nine weightlifting-trained men performed 15 sets of three repetitions of a dynamic clean pull exercise at 85% of their one repetition maximum. Vastus lateralis biopsies were obtained prior to (pre) and after the 8th (mid) and 15th set (post) of exercise. Three subjects returned to serve as non-exercising controls for a similar sequence of biopsies (CON). The ratio of phosphorylated MAPK to total MAPK increased significantly for p38 (3.0 fold, p < 0.05) and JNK (2.4 fold, p < 0.05) by the mid biopsy. ERK 1/2 phosphorylation followed a similar trend (2.3 fold) (p = 0.052). The ratio of phosphorylation to total MAPK did not differ from mid to post biopsy. None of the pathways were phosphorylated above resting in the CON condition (p > 0.05), and thus the biopsy procedure itself did not account for the entire increase in MAPK phosphorylation during EX. These data indicate MAPK pathways are activated early and remain elevated throughout the duration of high power resistance exercise. These findings help describe the mechanisms partially responsible for chronic adaptations in response to high intensity, high power resistance training in humans.

Pulse wave velocity and age- and gender-dependent aortic wall hardening in fowl.

Before sexual maturation, chickens (Gallus gallus) show high blood pressure (BP) and neointimal plaques in the lower abdominal aortae (AbA). We investigated age/sex-related changes in pulse wave velocity (PWV), elastin, collagen, and protein levels in AbA, and cardiac morphology to determine whether PWV increases during incremental increases in BP of maturing fowl, while arterial stiffness becomes dominant with aging. PWV (m/s) was significantly greater in male chicks (6-7 weeks, 9.3+/-0.8; females, 6.1+/-0.5) and remained high in cockerels (13 weeks), young (27-28 weeks), and adults (44-66 weeks). PWV increased in prepubertal pullets (10.0+/-0.9), dropped significantly in young hens, and remained low in adults. In contrast, medial thickness, protein levels, and collagen levels increased, while elastin/collagen ratios decreased, with maturation/aging. Males had heavier ventricular mass and thicker ventricular walls than females at all ages; left ventricular thickness decreased with maturation/aging. Thus, sustained high BP may have caused progressive medial hypertrophy, increased aortic rigidity, and enlarged hearts with left ventricular dilation. PWV of AbA was already greater in male chicks at an age when both sexes have similar collagen levels and low protein levels, suggesting that a factor other than structural stiffness may be an important determinant of PWV.

Laminin-induced activation of Rac1 and JNKp46 is initiated by Src family kinases and mimics the effects of skeletal muscle contraction.

Binding of laminin to dystroglycan in the dystrophin glycoprotein complex causes signaling through dystroglycan-syntrophin-grb2-SOS1-Rac1-PAK1-JNK. Laminin binding also causes syntrophin tyrosine phosphorylation to initiate signaling. The kinase responsible was investigated here. PP2 and SU6656, specific inhibitors of Src family kinases, decreased the amount of phosphotyrosine syntrophin and decreased the level of active Rac1 in laminin-treated myoblasts, myotubes, or skeletal muscle microsomes. c-Src and c-Fyn both phosphorylate syntrophin, and inhibition of either with specific siRNAs diminishes the level of syntrophin phosphorylation. When the rat gastrocnemius was contracted, the level of Rac1 activation increased compared to that of the relaxed control muscle and Rac1 colocalized with beta-dystroglycan. Similar results were obtained when the muscle was stretched. Contracted muscle also contained more activated c-Jun N-terminal kinase, JNKp46. E3, an expressed protein containing only laminin domains LG4 and LG5, increased the rate of proliferation of myoblasts, and PP2 prevented cell proliferation. In addition, Src family kinases colocalized with activated Rac1 and with laminin-Sepharose in solid-phase binding assays. Thus, contraction, stretching, or laminin binding causes recruitment of Src family kinase to the dystrophin glycoprotein complex, activating Rac1 and inducing downstream signaling. The DGC likely represents a mechanoreceptor in skeletal muscle-regulating muscle growth in response to muscle activity. Src family kinases play an initiating and critical role.

Ketosis-prone type 2 diabetes: effect of hyperglycemia on beta-cell function and skeletal muscle insulin signaling.

OBJECTIVE: To determine the underlying mechanism for the severe and transient beta-cell dysfunction and impaired insulin action in obese African American patients with ketosis-prone diabetes. METHODS: The effect of sustained hyperglycemia (glucotoxicity) and increased free fatty acids (lipotoxicity) on beta-cell function was assessed by changes in insulin secretion during a 20-hour glucose (200 mg/m2 per minute) and a 48-hour Intralipid (40 mL/h) infusion, respectively. Insulin-activated signaling pathways and pattern of Akt-1 and Akt-2 expression and insulin-stimulated phosphorylation were analyzed in skeletal muscle biopsy specimens. Studies were performed in an obese African American woman within 48 hours after resolution of diabetic ketoacidosis and 1 week after discontinuation of insulin treatment. RESULTS: Dextrose infusion rapidly increased C-peptide levels from a baseline of 3.2 ng/mL to a mean of 7.1 +/- 0.5 ng/mL during the first 8 hours of infusion; thereafter, C-peptide levels progressively declined. Lipid infusion was not associated with any deleterious effect on insulin and C-peptide secretion. Initial in vitro stimulation of muscle tissue with insulin resulted in a substantial and selectively decreased Akt-2 expression and insulin-stimulated phosphorylation on the serine residue. Improved metabolic control resulted in 70% greater Akt expression at near-normoglycemic remission in comparison with the period of hyperglycemia. CONCLUSION: Hyperglycemia, but not increased free fatty acid levels, led to progressive beta-cell dysfunction and impaired insulin secretion. Hyperglycemia was also associated with diminished skeletal muscle Akt expression and phosphorylation in an African American woman with ketosis-prone diabetes, and this defect improved notably with aggressive insulin therapy. These results indicate the importance of glucose toxicity in the pathogenesis of ketosis-prone diabetes in obese African American patients.

Binding of laminin alpha1-chain LG4-5 domain to alpha-dystroglycan causes tyrosine phosphorylation of syntrophin to initiate Rac1 signaling.

Previously, a signaling pathway was described [Oak, Zhou, and Jarrett (2003) J. Biol. Chem. 278, 39287-39295] that links matrix laminin binding on the outside of the sarcolemma to Grb2 binding to syntrophin on the inside surface of the sarcolemma and by way of Grb2-Sos1-Rac1-PAK1-JNK ultimately results in the phosphorylation of c-jun on Ser(65). How this signaling is initiated was investigated. Grb2-binding to syntrophin is increased by the addition of either laminin-1 or the isolated laminin alpha1 globular domain modules LG4-5, a protein referred to as E3. This identifies the LG4-5 sequences as the region of laminin responsible for signaling. Since laminin alpha1 LG4 is known to bind alpha-dystroglycan, this directly implicates alpha-dystroglycan as the laminin-signaling receptor. E3 or laminin-1 increase Grb2-binding and Rac1 activation. In the presence of E3 or laminin-1, syntrophin is phosphorylated on a tyrosine residue, and this increases and alters Grb2 binding. The alpha-dystroglycan antibody, IIH6, which blocks binding of laminins to alpha-dystroglycan, blocks both the laminin-induced Sos1/2 recruitment and syntrophin phosphorylation, showing that it is alpha-dystroglycan binding the LG4-5 region of laminin that is responsible. The C-terminal SH3 domain of Grb2 (C-SH3) binds only to nonphosphorylated syntrophin, and phosphorylation causes the Grb2 SH2 domain to bind and prevents SH3 binding. Syntrophin, tyrosine phosphate, beta-dystroglycan, and Rac1 all co-localize to the sarcolemma of rat muscle sections. A model for how this phosphorylation may initiate downstream events in laminin signaling is presented.

Impaired expression and insulin-stimulated phosphorylation of Akt-2 in muscle of obese patients with atypical diabetes.

Although a pharmacological dose of insulin produces a dramatic increase in phosphorylation and activity of Akt isoforms 1 and 2 in mammalian skeletal muscle, few studies have examined the effect of physiological concentrations of insulin on the phosphorylation of Akt-1 and -2 in normal and diabetic tissue. This study examined the patterns of insulin-stimulated Akt isoform phosphorylation and protein expression in muscle biopsies obtained from obese patients with atypical diabetes immediately after a hyperglycemic crisis and again after near-normoglycemic remission. In obese patients with new-onset diabetes mellitus presenting with hyperglycemic crisis (plasma glucose 30.5 +/- 4.8 mM), in vitro stimulation of vastus lateralis muscle biopsies with 100 microU/ml (0.6 nM) insulin increased insulin receptor phosphorylation threefold and Akt-1 phosphorylation on Ser(473) twofold, whereas Akt-2 phosphorylation was not stimulated. After 10-wk intensive insulin therapy that led to near-normoglycemic remission and discontinuation of insulin therapy, both Akt-2 expression and insulin-stimulated Akt-2 Ser(474) phosphorylation doubled. Hyperglycemic crisis did not affect insulin-stimulated threonine phosphorylation of either Akt-1 or Akt-2. The decreased Akt-2 expression at presentation was accompanied by reduced GLUT4 protein expression and increased expression of enzymes counterregulatory to insulin action. Thus a physiological concentration of insulin stimulated Akt-1 and Akt-2 phosphorylation in human skeletal muscle in the absence of hyperglycemia, but Akt-2 expression and stimulation appeared to be impaired in muscle of obese patients with atypical diabetes presenting with severe hyperglycemia.

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle.

In mildly hyperosmotic medium, activation of the Na+ -K+ -2Cl- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (KATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a KATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of KATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of KATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions.

NKCC activity restores muscle water during hyperosmotic challenge independent of insulin, ERK, and p38 MAPK.

In isosmotic conditions, insulin stimulation of PI 3-K/Akt and p38 MAPK pathways in skeletal muscle inhibits Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity induced by the ERK1,2 MAPK pathway. Whether these signaling cascades contribute to NKCC regulation during osmotic challenge is unknown. Increasing osmolarity by 20 mosM with either glucose or mannitol induced NKCC-mediated (86)Rb uptake and water transport into rat soleus and plantaris skeletal muscle in vitro. This NKCC activity restored intracellular water. In contrast to mannitol, hyperosmolar glucose increased ERK1,2 and p38 MAPK phosphorylation. Glucose, but not mannitol, impaired insulin-stimulated phosphorylation of Akt and p38 MAPK in the plantaris and soleus muscles, respectively. Hyperosmolarity-induced NKCC activation was insensitive to insulin action and pharmacological inhibition of ERK1,2 and p38 MAPK pathways. Paradoxically, cAMP-producing agents, which stimulate NKCC activity in isosmotic conditions, suppressed hyperosmolar glucose- and mannitol-induced NKCC activity and prevented restoration of muscle cell volume in hyperosmotic media. These results indicate that NKCC activity helps restore muscle cell volume during hyperglycemia. Moreover, hyperosmolarity activates NKCC regulatory pathways that are insensitive to insulin inhibition.

Exercise effects on muscle beta-adrenergic signaling for MAPK-dependent NKCC activity are rapid and persistent.

This study investigated exercise adaptation of signaling mechanisms that control Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity in rat skeletal muscle. An acute bout of exercise increased total and NKCC-mediated (86)Rb influx. Inhibition of extracellular signal-regulated kinase (ERK) activation abolished the exercise-induced NKCC upregulation. Treadmill training (20 m/min, 20% grade, 30 min/day, 5 days/wk) stimulated total (86)Rb influx and increased NKCC activity in the soleus muscle after 2 wk and in the plantaris muscle after 4 wk. Exercise-induced NKCC activity was associated with a 1.4- to 2-fold increase in ERK phosphorylation. Isoproterenol, which activates ERK and NKCC in sedentary muscle, caused a remarkable inhibition of the exercise-induced NKCC activity. Furthermore, isoproterenol inhibition of exercise-induced NKCC activity was accompanied with decreased ERK phosphorylation in the plantaris muscle. Akt (protein kinase B) phosphorylation on both Thr(308) and Ser(473), which activates Akt and inhibits NKCC activity in sedentary muscle, was stimulated by acute and chronic exercise. This Akt activation was unaffected by isoproterenol. These results indicate an immediate and persistent exercise adaptation of the signal pathways that participate in the control of potassium transport.

Duality of G protein-coupled mechanisms for beta-adrenergic activation of NKCC activity in skeletal muscle.

Skeletal muscle Na(+)-K(+)-2Cl(-) cotransporter (NKCC) activity provides a potential mechanism for regulated K(+) uptake. beta-Adrenergic receptor (beta-AR) activation stimulates skeletal muscle NKCC activity in a MAPK pathway-dependent manner. We examined potential G protein-coupled pathways for beta-AR-stimulated NKCC activity. Inhibition of G(s)-coupled PKA blocked isoproterenol-stimulated NKCC activity in both the slow-twitch soleus muscle and the fast-twitch plantaris muscle. However, the PKA-activating agents cholera toxin, forskolin, and 8-bromo-cAMP (8-BrcAMP) were not sufficient to activate NKCC in the plantaris and partially stimulated NKCC activity in the soleus. Isoproterenol-stimulated NKCC activity in the soleus was abolished by pretreatment with pertussis toxin (PTX), indicating a G(i)-coupled mechanism. PTX did not affect the 8-BrcAMP-stimulated NKCC activity. PTX treatment also precluded the isoproterenol-mediated ERK1/2 MAPK phosphorylation in the soleus, consistent with NKCC's MAPK dependency. Inhibition of isoproterenol-stimulated ERK activity by PTX treatment was associated with an increase in Akt activation and phosphorylation of Raf-1 on the inhibitory residue Ser(259). These results demonstrate a novel, muscle phenotype-dependent mechanism for beta-AR-mediated NKCC activation that involves both G(s) and G(i) protein-coupled mechanisms.

Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy.

The dystrophin-glycoprotein complex (DGC) is a sarcolemmal complex whose defects cause muscular dystrophies. The normal function of this complex is not clear. We have proposed that this is a signal transduction complex, signaling normal interactions with matrix laminin, and that the response is normal growth and homeostasis. If so, the complex and its signaling should be altered in other physiological states such as atrophy. The amount of some of the DGC proteins, including dystrophin, beta-dystroglycan, and alpha-sarcoglycan, is reduced significantly in rat skeletal muscle atrophy induced by tenotomy. Furthermore, H-Ras, RhoA, and Cdc42 decrease in expression levels and activities in muscle atrophy. When the small GTPases were assayed after laminin or beta-dystroglycan depletion, H-Ras, Rac1, and Cdc42 activities were reduced, suggesting a physical linkage between the DGC and the GTPases. Dominant-negative Cdc42, introduced with a retroviral vector, resulted in fibers that appeared atrophic. These data support a putative role for the DGC in transduction of mechanical signals in muscle.

Insulin and isoproterenol differentially regulate mitogen-activated protein kinase-dependent Na(+)-K(+)-2Cl(-) cotransporter activity in skeletal muscle.

Recent studies have demonstrated that p44/42(MAPK) extracellular signal-regulated kinase (ERK)1 and -2-dependent Na(+)-K(+)-2Cl(-) co-transporter (NKCC) activity may contribute to total potassium uptake by skeletal muscle. To study the precise mechanisms regulating NKCC activity, rat soleus and plantaris muscles were stimulated ex vivo by insulin or isoproterenol (ISO). Both hormones stimulated total uptake of the potassium congener (86)Rb by 25--70%. However, only ISO stimulated the NKCC-mediated (86)Rb uptake. Insulin inhibited the ISO-stimulated NKCC activity, and this counteraction was sensitive to the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 in the predominantly slow-twitch soleus muscle. Pretreatment of the soleus muscle with the phosphatidylinositol (PI) 3-kinase inhibitors wortmannin and LY294002 or with SB203580 uncovered an insulin-stimulated NKCC activity and also increased the insulin-stimulated phosphorylation of ERK. In the predominantly fast-twitch plantaris muscle, insulin-stimulated NKCC activity became apparent only after inhibition of PI 3-kinase activity, accompanied by an increase in ERK phosphorylation. PI 3-kinase inhibitors also abolished insulin-stimulated p38 MAPK phosphorylation in the plantaris muscle and Akt phosphorylation in both muscles. These data demonstrated that insulin inhibits NKCC-mediated transport in skeletal muscle through PI 3-kinase-sensitive and SB203580-sensitive mechanisms. Furthermore, differential activation of signaling cascade elements after hormonal stimulation may contribute to fiber-type specificity in the control of potassium transport by skeletal muscle.