E-Tattoos: Toward Functional but Imperceptible Interfacing with Human Skin.

The human body continuously emits physiological and psychological information from head to toe. Wearable electronics capable of noninvasively and accurately digitizing this information without compromising user comfort or mobility have the potential to revolutionize telemedicine, mobile health, and both human-machine or human-metaverse interactions. However, state-of-the-art wearable electronics face limitations regarding wearability and functionality due to the mechanical incompatibility between conventional rigid, planar electronics and soft, curvy human skin surfaces. E-Tattoos, a unique type of wearable electronics, are defined by their ultrathin and skin-soft characteristics, which enable noninvasive and comfortable lamination on human skin surfaces without causing obstruction or even mechanical perception. This review article offers an exhaustive exploration of e-tattoos, accounting for their materials, structures, manufacturing processes, properties, functionalities, applications, and remaining challenges. We begin by summarizing the properties of human skin and their effects on signal transmission across the e-tattoo-skin interface. Following this is a discussion of the materials, structural designs, manufacturing, and skin attachment processes of e-tattoos. We classify e-tattoo functionalities into electrical, mechanical, optical, thermal, and chemical sensing, as well as wound healing and other treatments. After discussing energy harvesting and storage capabilities, we outline strategies for the system integration of wireless e-tattoos. In the end, we offer personal perspectives on the remaining challenges and future opportunities in the field.

Role of Hepatic Stellate and Liver Sinusoidal Endothelial Cells in a Human Primary Cell Three-Dimensional Model of Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is an inflammatory and fibrotic liver disease that has reached epidemic proportions and has no approved pharmacologic therapies. Research and drug development efforts are hampered by inadequate preclinical models. This research describes a three-dimensional bioprinted liver tissue model of NASH built using primary human hepatocytes and nonparenchymal liver cells (hepatic stellate cells, liver sinusoidal endothelial cells, and Kupffer cells) from either healthy or NASH donors. Three-dimensional tissues bioprinted with cells sourced from diseased patients showed a NASH phenotype, including fibrosis. More importantly, this NASH phenotype occurred without the addition of disease-inducing agents. Bioprinted tissues composed entirely of healthy cells exhibited significantly less evidence of disease. The role of individual cell types in driving the NASH phenotype was examined by producing chimeric bioprinted tissues composed of healthy cells together with the addition of one or more diseased nonparenchymal cell types. These experiments reveal a role for both hepatic stellate and liver sinusoidal endothelial cells in the disease process. This model represents a fully human system with potential to detect clinically active targets and eventually therapies.

Merging DNA Repair with Bioorthogonal Conjugation Enables Accessible and Versatile Asymmetric DNA Catalysis.

Optimizing catalysts through high-throughput screening for asymmetric catalysis is challenging due to the difficulty associated with assembling a library of catalyst analogues in a timely fashion. Here, we repurpose DNA excision repair and integrate it with bioorthogonal conjugation to construct a diverse array of DNA hybrid catalysts for highly accessible and high-throughput asymmetric DNA catalysis, enabling a dramatically expedited catalyst optimization process, superior reactivity and selectivity, as well as the first atroposelective DNA catalysis. The bioorthogonality of this conjugation strategy ensures exceptional tolerance toward diverse functional groups, thereby facilitating the facile construction of 44 DNA hybrid catalysts bearing various unprotected functional groups. This unique feature holds the potential to enable catalytic modalities in asymmetric DNA catalysis that were previously deemed unattainable.

Differential sensitivity to longitudinal and transverse stretch mediates transcriptional responses in mouse neonatal ventricular myocytes.

To identify how cardiomyocyte mechanosensitive signaling pathways are regulated by anisotropic stretch, micropatterned mouse neonatal cardiomyocytes were stretched primarily longitudinally or transversely to the myofiber axis. Four hours of static, longitudinal stretch induced differential expression of 557 genes, compared with 30 induced by transverse stretch, measured using RNA-seq. A logic-based ordinary differential equation model of the cardiac myocyte mechanosignaling network, extended to include the transcriptional regulation and expression of 784 genes, correctly predicted measured expression changes due to anisotropic stretch with 69% accuracy. The model also predicted published transcriptional responses to mechanical load in vitro or in vivo with 63-91% accuracy. The observed differences between transverse and longitudinal stretch responses were not explained by differential activation of specific pathways but rather by an approximately twofold greater sensitivity to longitudinal stretch than transverse stretch. In vitro experiments confirmed model predictions that stretch-induced gene expression is more sensitive to angiotensin II and endothelin-1, via RhoA and MAP kinases, than to the three membrane ion channels upstream of calcium signaling in the network. Quantitative cardiomyocyte gene expression differs substantially with the axis of maximum principal stretch relative to the myofilament axis, but this difference is due primarily to differences in stretch sensitivity rather than to selective activation of mechanosignaling pathways.NEW & NOTEWORTHY Anisotropic stretch applied to micropatterned neonatal mouse ventricular myocytes induced markedly greater acute transcriptional responses when the major axis of stretch was parallel to the myofilament axis than when it was transverse. Analysis with a novel quantitative network model of mechanoregulated cardiomyocyte gene expression suggests that this difference is explained by higher cell sensitivity to longitudinal loading than transverse loading than by the activation of differential signaling pathways.

Benchmarking of protein interaction databases for integration with manually reconstructed signalling network models.

Protein interaction databases are critical resources for network bioinformatics and integrating molecular experimental data. Interaction databases may also enable construction of predictive computational models of biological networks, although their fidelity for this purpose is not clear. Here, we benchmark protein interaction databases X2K, Reactome, Pathway Commons, Omnipath and Signor for their ability to recover manually curated edges from three logic-based network models of cardiac hypertrophy, mechano-signalling and fibrosis. Pathway Commons performed best at recovering interactions from manually reconstructed hypertrophy (137 of 193 interactions, 71%), mechano-signalling (85 of 125 interactions, 68%) and fibroblast networks (98 of 142 interactions, 69%). While protein interaction databases successfully recovered central, well-conserved pathways, they performed worse at recovering tissue-specific and transcriptional regulation. This highlights a knowledge gap where manual curation is critical. Finally, we tested the ability of Signor and Pathway Commons to identify new edges that improve model predictions, revealing important roles of protein kinase C autophosphorylation and Ca2+ /calmodulin-dependent protein kinase II phosphorylation of CREB in cardiomyocyte hypertrophy. This study provides a platform for benchmarking protein interaction databases for their utility in network model construction, as well as providing new insights into cardiac hypertrophy signalling. KEY POINTS: Protein interaction databases are used to recover signalling interactions from previously developed network models. The five protein interaction databases benchmarked recovered well-conserved pathways, but did poorly at recovering tissue-specific pathways and transcriptional regulation, indicating the importance of manual curation. We identify new signalling interactions not previously used in the network models, including a role for Ca2+ /calmodulin-dependent protein kinase II phosphorylation of CREB in cardiomyocyte hypertrophy.

Predictive model identifies key network regulators of cardiomyocyte mechano-signaling.

Mechanical strain is a potent stimulus for growth and remodeling in cells. Although many pathways have been implicated in stretch-induced remodeling, the control structures by which signals from distinct mechano-sensors are integrated to modulate hypertrophy and gene expression in cardiomyocytes remain unclear. Here, we constructed and validated a predictive computational model of the cardiac mechano-signaling network in order to elucidate the mechanisms underlying signal integration. The model identifies calcium, actin, Ras, Raf1, PI3K, and JAK as key regulators of cardiac mechano-signaling and characterizes crosstalk logic imparting differential control of transcription by AT1R, integrins, and calcium channels. We find that while these regulators maintain mostly independent control over distinct groups of transcription factors, synergy between multiple pathways is necessary to activate all the transcription factors necessary for gene transcription and hypertrophy. We also identify a PKG-dependent mechanism by which valsartan/sacubitril, a combination drug recently approved for treating heart failure, inhibits stretch-induced hypertrophy, and predict further efficacious pairs of drug targets in the network through a network-wide combinatorial search.

In vitro comparison of guided versus freehand implant placement: use of a new combined TRIOS surface scanning, Implant Studio, CBCT, and stereolithographic virtually planned and guided technique.

Implant placement requires precise planning and execution to avoid collision with critical anatomical structures. Technology advances may improve placement outcomes. The purpose of this study was to trial and measure in an in vitro environment the accuracy of placing a single dental implant in the planned position using a specific guided surgery technique compared with a freehand surgery technique. The dental model of a patient missing tooth 16 was printed 30 times (EnvisionTEC 3Dent). Each print was scanned (TRIOS color scanner) to create a 3D surface model, and radiographed (Gendex CB-500) to create cone beam computed tomography (CBCT) data. The surface data and CBCT data were merged (Implant Studio software), and a Straumann RC bone level Ø 4.1 × 8 mm implant placement was planned. A surgical guide was printed (Stratasys OrthoDesk) for each case (n = 30). Simulated cases were assigned to Group A (guided) or Group B (freehand, where the fabricated guide was discarded). Implants were placed, and the models rescanned (TRIOS). The new data was superimposed on the original data, and the surgical implant location compared with the planned position for each model (Convince software) by a researcher blinded to group allocation. Differences in angulation (degrees); shoulder, apex, and depth displacements (mm); and direction of displacement were assessed with Mann-Whitney U and Fisher exact tests. Data was expressed as medians bounded by interquartile ranges (IQRs). Implant angulation and apical displacement were significantly closer to the planned position in the guided group compared with the freehand group (3.91 degrees: IQR 2.45 to 5.38 degrees vs 8.82 degrees: IQR 4.84 to 9.84 degrees, P = 0.005; and 0.87 mm: IQR 0.53 to 1.11 mm vs 1.48 mm: IQR 1.14 to 1.72 mm, P < 0.001, respectively). Implant shoulder displacement, depth displacements, and direction of displacement did not differ between the groups. Within the in vitro environment, merged 3D surface scan data and 3D CBCT scan data can be used to plan and guide implant placement with greater accuracy than with the freehand technique.

Coevolution of URAT1 and Uricase during Primate Evolution: Implications for Serum Urate Homeostasis and Gout.

Uric acid is the highly insoluble end-product of purine metabolism in humans. Serum levels exceeding the solubility threshold can trigger formation of urate crystals resulting in gouty arthritis. Uric acid is primarily excreted through the kidneys with 90% reabsorbed back into the bloodstream through the uric acid transporter URAT1. This reabsorption process is essential for the high serum uric acid levels found in humans. We discovered that URAT1 proteins from humans and baboons have higher affinity for uric acid compared with transporters from rats and mice. This difference in transport kinetics of URAT1 orthologs, along with inability of modern apes to oxidize uric acid due to loss of the uricase enzyme, prompted us to ask whether these events occurred concomitantly during primate evolution. Ancestral URAT1 sequences were computationally inferred and ancient transporters were resurrected and assayed, revealing that affinity for uric acid was increased during the evolution of primates. This molecular fine-tuning occurred between the origins of simians and their diversification into New- and Old-World monkey and ape lineages. Remarkably, it was driven in large-part by only a few amino acid replacements within the transporter. This alteration in primate URAT1 coincided with changes in uricase that greatly diminished the enzymatic activity and took place 27-77 Ma. These results suggest that the modifications to URAT1 transporters were potentially adaptive and that maintaining more constant, high levels of serum uric acid may have provided an advantage to our primate ancestors.

Similarities and Differences in Pacing Patterns in a 161-km and 101-km Ultra-Distance Road Race.

Tan, PLS, Tan, FHY, and Bosch, AN. Similarities and differences in pacing patterns in a 161-km and 101-km ultra-distance road race. J Strength Cond Res 30(8): 2145-2155, 2016-The purpose of this study was to establish and compare the pacing patterns of fast and slow finishers in a tropical ultra-marathon. Data were collected from the Craze Ultra-marathon held on the 22nd and 21st of September in 2012 and 2013, respectively. Finishers of the 161-km (N = 47) and 101-km (N = 120) categories of the race were divided into thirds (groups A-C) by merit of finishing time. Altogether, 17 and 11 split times were recorded for the 161-km and 101-km finishers, respectively, and used to calculate the mean running speed for each distance segment. Running speed for the first segment was normalized to 100, with all subsequent splits adjusted accordingly. Running speed during the last 5 km was calculated against the mean race pace to establish the existence of an end spurt. A reverse J-shaped pacing profile was demonstrated in all groups for both distance categories and only 38% of the finishers executed an end spurt. In the 101-km category, in comparison with groups B and C, group A maintained a significantly more even pace (p = 0.013 and 0.001, respectively) and completed the race at a significantly higher percent of initial starting speed (p = 0.001 and 0.001, respectively). Descriptive data also revealed that the top 5 finishers displayed a "herd-behavior" by staying close to the lead runner in the initial portion of the race. These findings demonstrate that to achieve a more even pace, recreational ultra-runners should adopt a patient sustainable starting speed, with less competitive runners setting realistic performance goals whereas competitive runners with a specific time goal to consider running in packs of similar pace.

Towards Simultaneous Noninvasive Arterial and Venous Oxygenation Monitoring with Wearable E-Tattoo.

While noninvasive arterial blood oxygenation is easily estimated using peripheral pulse oximeters, noninvasive venous blood oxygenation monitoring is still a critical unmet need. Critical conditions that lead to inefficient extraction of oxygen from the blood, such as sepsis or shock, can only be detected by analyzing the oxygen content of the venous blood. In this work, we introduce a soft wearable e-tattoo sensor that simultaneously measures the arterial and venous pulses from the wrist. First, we prove that the origin of the signal is venous pulsatility. We hypothesize that a significant obstacle for simultaneous SaO2 and SvO2 extraction is the close proximity of the artery and vein, thus leading to crosstalk. We characterize this crosstalk with simulation, in vitro, and in vivo experiments. Finally, we offer a potential solution for minimizing the crosstalk through spatial filtering.Clinical Relevance- This lays foundational work for a novel method of noninvasively and simultaneously measuring arterial and venous blood oxygenation to improve clinical diagnoses of sepsis, shock, and metabolic abnormalities.

Seeing inside a body in motion.

Adhesive ultrasound patches can provide medical imaging for patients on the go.

Salmonella Mississippi: An unusual cause of renal abscess in an immunocompetent patient.

Renal abscess is a rare manifestation of Salmonella infection. This usually occurs in the presence of risk factors that include immunosuppression, renal stones and urinary structural abnormality. We describe a 19-year-old male with no risk factors who developed a left renal abscess and gram-negative sepsis caused by Salmonella Mississippi. This was managed successfully with percutaneous drainage of the abscess and a prolonged course of antibiotics. To our knowledge, this is the first reported case of Salmonella Mississippi as a cause for renal abscess in an individual with no identifiable risk factors.

Highly Sensitive Capacitive Pressure Sensors over a Wide Pressure Range Enabled by the Hybrid Responses of a Highly Porous Nanocomposite.

Past research aimed at increasing the sensitivity of capacitive pressure sensors has mostly focused on developing dielectric layers with surface/porous structures or higher dielectric constants. However, such strategies have only been effective in improving sensitivities at low pressure ranges (e.g., up to 3 kPa). To overcome this well-known obstacle, herein, a flexible hybrid-response pressure sensor (HRPS) composed of an electrically conductive porous nanocomposite (PNC) laminated with an ultrathin dielectric layer is devised. Using a nickel foam template, the PNC is fabricated with carbon nanotubes (CNTs)-doped Ecoflex to be 86% porous and electrically conductive. The PNC exhibits hybrid piezoresistive and piezocapacitive responses, resulting in significantly enhanced sensitivities (i.e., more than 400%) over wide pressure ranges, from 3.13 kPa-1 within 0-1 kPa to 0.43 kPa-1 within 30-50 kPa. The effect of the hybrid responses is differentiated from the effect of porosity or high dielectric constants by comparing the HRPS with its purely piezocapacitive counterparts. Fundamental understanding of the HRPS and the prediction of optimal CNT doping are achieved through simplified analytical models. The HRPS is able to measure pressures from as subtle as the temporal arterial pulse to as large as footsteps.

CD20-directed small modular immunopharmaceutical, TRU-015, depletes normal and malignant B cells.

PURPOSE: CD20-directed therapy with rituximab is effective in many patients with malignant lymphoma or follicular lymphoma. However, relapse frequently occurs within 1 year, and patients become increasingly refractory to retreatment. Our purpose was to produce a compact, single-chain CD20-targeting immunotherapeutic that could offer therapeutic advantages in the treatment of B-cell lymphoma. EXPERIMENTAL DESIGN: Rituximab is a chimeric antibody containing two heavy chains and two light chains. Here, we describe the properties of TRU-015, a small modular immunopharmaceutical specific for CD20, encoded by a single-chain construct containing a single-chain Fv specific for CD20 linked to human IgG1 hinge, CH2, and CH3 domains but devoid of CH1 and CL domains. RESULTS: TRU-015 mediates potent direct signaling and antibody-dependent cellular cytotoxicity but has reduced size and complement-mediated cytotoxicity activity compared with rituximab. TRU-015 is a compact dimer of 104 kDa that comigrates with albumin in size exclusion chromatography and retains a long half-life in vivo. TRU-015 induced growth arrest in multiple B lymphoma cell lines in vitro and showed effective antitumor activity against large, established subcutaneous Ramos or Daudi xenograft tumors in nude mice. TRU-015 also showed rapid, dose-dependent, and durable depletion of peripheral blood B cells following single-dose administration to nonhuman primates. CONCLUSION: These results indicate that TRU-015 may improve CD20-directed therapy by effectively depleting embedded malignant B cells and nonmalignant pathogenic B cells and do so with reduced complement activation.

Mechanical regulation of gene expression in cardiac myocytes and fibroblasts.

The intact heart undergoes complex and multiscale remodelling processes in response to altered mechanical cues. Remodelling of the myocardium is regulated by a combination of myocyte and non-myocyte responses to mechanosensitive pathways, which can alter gene expression and therefore function in these cells. Cellular mechanotransduction and its downstream effects on gene expression are initially compensatory mechanisms during adaptations to the altered mechanical environment, but under prolonged and abnormal loading conditions, they can become maladaptive, leading to impaired function and cardiac pathologies. In this Review, we summarize mechanoregulated pathways in cardiac myocytes and fibroblasts that lead to altered gene expression and cell remodelling under physiological and pathophysiological conditions. Developments in systems modelling of the networks that regulate gene expression in response to mechanical stimuli should improve integrative understanding of their roles in vivo and help to discover new combinations of drugs and device therapies targeting mechanosignalling in heart disease.

Ribozyme technology for cancer gene target identification and validation.

Ribozymes are naturally occurring RNAs with catalytic activities including cis- or trans- cleavage of RNA at predefined sequence sites. This activity has been exploited for specific gene inactivation in cells during the last two decades, and ribozymes have been important functional genomics tools, especially in the pre-RNAi era. It has also been broadly applied in drug target identification and validation in pharmaceutical R&D. This chapter covers many application principles and case studies of ribozyme technology in the areas of cancer research. We also described RNAi applications in some of the same studies for comparison. Although RNAi may be more effective than ribozymes in many respects, they are nonetheless built on many of the same principles.

An in vitro comparison of vertical marginal gaps of CAD/CAM titanium and conventional cast restorations.

PURPOSE: To determine if there was a significant difference between the vertical marginal openings of cast restorations, computer-aided design, and computer-aided machining restorations. MATERIALS AND METHODS: Ten working dies were created from a single master die and used to fabricate ten restorations in each of the following groups: computer-aided design/computer-assisted machining (CAD/CAM), WAX/CAM, and WAX/CAST. The CAD/CAM titanium restorations were fabricated using the scanning and crown design modules of the KaVo Everest system. The WAX/CAM titanium restorations were fabricated using the double scan technique with the KaVo Everest system. The WAX/CAST high noble copings were fabricated using the conventional lost wax casting technique. The restorations were seated on the master die, and high-resolution digital photographs were made of the marginal area on all four sides. The vertical marginal opening was then measured using a calibrated digital software program. One-way ANOVA and Tukey's post hoc tests were used to determine the presence of statistically significant differences. RESULTS: The vertical margin openings were CAD/CAM: 79.43 +/- 25.46 microm; WAX/CAM: 73.12 +/- 24.15 microm; WAX/CAST: 23.91 +/- 9.80 microm. There was a statistically significant difference between the WAX/CAST group and the remaining groups. CONCLUSIONS: There was no difference between the vertical marginal gaps of the CAD/CAM and WAX/CAM. The WAX/CAST technique resulted in smaller vertical marginal gaps than either CAD/CAM or WAX/CAM.

High content analysis identifies unique morphological features of reprogrammed cardiomyocytes.

Direct reprogramming of fibroblasts into cardiomyocytes is a promising approach for cardiac regeneration but still faces challenges in efficiently generating mature cardiomyocytes. Systematic optimization of reprogramming protocols requires scalable, objective methods to assess cellular phenotype beyond what is captured by transcriptional signatures alone. To address this question, we automatically segmented reprogrammed cardiomyocytes from immunofluorescence images and analyzed cell morphology. We also introduce a method to quantify sarcomere structure using Haralick texture features, called SarcOmere Texture Analysis (SOTA). We show that induced cardiac-like myocytes (iCLMs) are highly variable in expression of cardiomyocyte markers, producing subtypes that are not typically seen in vivo. Compared to neonatal mouse cardiomyocytes, iCLMs have more variable cell size and shape, have less organized sarcomere structure, and demonstrate reduced sarcomere length. Taken together, these results indicate that traditional methods of assessing cardiomyocyte reprogramming by quantifying induction of cardiomyocyte marker proteins may not be sufficient to predict functionality. The automated image analysis methods described in this study may enable more systematic approaches for improving reprogramming techniques above and beyond existing algorithms that rely heavily on transcriptome profiling.

Monitoring interactions between receptor tyrosine kinases and their downstream effector proteins in living cells using bioluminescence resonance energy transfer.

A limited number of whole-cell assays allow monitoring of receptor tyrosine kinase (RTK) activity in a signaling pathway-specific manner. We present the general use of the bioluminescence resonance energy transfer (BRET) technology to quantitatively study the pharmacology and signaling properties of the receptor tyrosine kinase (RTK) superfamily. RTK BRET-2 assays monitor, in living cells, the specific interaction between RTKs and their effector proteins, which control the activation of specific downstream signaling pathways. A total of 22 BRET assays have been established for nine RTKs derived from four subfamilies [erythroblastic leukemia viral (v-erb-b) oncogene homolog (ErbB), platelet-derived growth factor (PDGF), neurotrophic tyrosine kinase receptor (TRK), vascular endothelial growth factor (VEGF)] monitoring the interactions with five effectors (Grb2, p85, Stat5a, Shc46, PLCgamma1). These interactions are dependent on the RTK kinase activity and autophosphorylation of specific tyrosine residues in the carboxyl terminus. RTK BRET assays are highly sensitive for quantifying ligand-independent (constitutive), agonist-induced, or antagonist-inhibited RTK activity levels. We studied the signaling properties of the PDGF receptor, alpha polypeptide (PDGFRA) isoforms (V561D; D842V and delta842-845) carrying activating mutations identified in gastrointestinal stromal tumors (GIST). All three PDGFRA isoforms are fully constitutively activated, insensitive to the growth factor PDGF-BB, but show differential sensitivity of their constitutive activity to be inhibited by the inhibitor imatinib (Gleevec). Epidermal growth factor receptor (EGFR) BRET structure-function studies identify the tyrosine residues 1068, 1114, and 1148 as the main residues mediating the interaction of EGFR with the adapter protein Grb2. The BRET technology provides an assay platform to study signaling pathway-specific RTK structure-function and will facilitate drug discovery efforts for the identification of novel RTK modulators.

Assessment of Differences in the Anthropometric, Physiological and Training Characteristics of Finishers and Non-finishers in a Tropical 161-km Ultra-marathon.

This study aimed to compare and determine the differences in the physiological, anthropometric and training characteristics of the finishers (FIN) and non-finishers (N-FIN) in a 161-km race. Two groups of runners (FIN; N=12 and N-FIN; N=14) completed a series of anthropometric and physiological measurements over two separate sessions at least three weeks prior to the race. Training sessions starting from six weeks prior to the race were recorded. Sum of 7 skinfolds, arm and calf girths, VO2max and peak treadmill speed (PTS) were taken during session 1 while the lactate threshold (LT) and running economy (RE) were assessed during session 2. Effect size calculations showed moderate and clear differences in the lactate concentration at LT1 (ES = 0.88, P = 0.05), velocity at LT2 (ES = 0.70, P = 0.07), longest run attempted (ES = 0.73, P = 0.07) and number of cross-training hours (ES = 0.73, P = 0.06) between the FIN and N-FIN. The results suggest that from a physiological perspective, the ability to finish a 161-km race might be differentiated by metabolic attributes via LT measurements. Runners should not neglect the importance of the long runs and should incorporate cross-training to provide additional stimuli to the body while allowing the running muscles to recover from fatigue.