The functions of organ systems must be coordinated for physiological homeostasis to occur. For example, after mating, coordination between insect digestive and reproductive systems is needed to ensure adequate nutrition for efficient egg/progeny production, and, conversely, to attune egg production levels to nutrient availability. Recent studies of Drosophila have revealed much about the post-mating changes in female reproductive tract function and in gut homeostasis, and the induction of these changes by male seminal proteins. Interesting regulatory connections between the organ systems and their responses have come to light in those studies. We have gathered these data into a single network schematic of the signaling events that operate within and between the reproductive and digestive systems downstream of seminal fluid proteins, summarizing current knowledge of the crosstalk between the systems and raising open questions for future study.Drosophila melanogaster, have described the mechanisms of post-mating changes within the female reproductive tract and digestive system. Additionally, other studies have described post-mating signaling crosstalk between these systems. Interestingly, male seminal fluid proteins have been linked to post-mating responses within the female reproductive tract and gut, and to signaling events between the two organ systems. However, information about the hormonal and neuronal signaling pathways underlying the post-mating signaling events within and between the reproductive tract and digestive systems that are triggered by seminal fluid proteins has yet to be combined into a single view. In this article, we summarize and integrate these studies into a single “network schematic” of the known signaling events within and between the reproductive and digestive systems downstream of male seminal fluid proteins. This synthesis also draws attention to the incomplete parts of these pathways, so that outstanding questions may be addressed in future studies. Mating initiates broad physiological changes encompassing multiple organ systems in females. Elucidating the complex inter- and intra-organ signaling events that coordinate these physiological changes is an important goal in the field of reproductive biology. Further characterization of these complex molecular and physiological interactions is key to understanding how females meet the energetic demands of offspring production. Many recent studies of the fruit fly,  Throughout the animal kingdom, female nutrition is connected to reproductive output. Females must consume enough appropriate nutrients to energetically support pregnancy   or the pDrosophila melanogaster makes it an excellent system for dissecting how changes within the digestive and reproductive systems, as well as crosstalk between these two organ systems, can affect offspring production. After mating, Drosophila melanogaster females undergo a suite of physiological and behavioral changes that transition the female from a virgin state to a highly fecund mated state. Collectively, these changes are referred to as the post-mating response (PMR). In the female reproductive tract , during mating. This 36-amino-acid peptide triggers transitions from a virgin to a mated state ,39,40,41+Tdc2, +dsx neurons that project to the female reproductive tract from the abdominal ganglion  activity in a subset of somatic cells in the germarium of the ovary , and BMogenesis  22]. Th. ThTdc2+Interactions between the SP–SPSN–OA axis and ovulin, another seminal fluid protein that affects OA signaling, should also be examined . Ovulin,SP–SPSN neuronal signaling also triggers physiological changes in the female reproductive tract via other neuronal circuits. An SP–SPR-regulated neuronal circuit containing the SPSNs, SAG neurons in the abdominal ganglion, pC1, and oviposition inhibitory (oviINs) neurons in the brain controls oviposition, in conjunction with oviposition descending neurons (oviDNs) that project from the brain to the abdominal ganglion  20]. Sp. Sp20]. SP–SPR signaling does not only trigger post-mating responses via the SPSNs. The SPR is also expressed in the oviduct and in the spermathecal secretory cells (SSCs) . While tHGN1-GAL4-positive neurons in the hindgut  axis. Under normal conditions, 20E stimulates ETH expression in Inka cells  and ETHR2+ and Mmp2. However, again, the details of this interaction are unknown  via the JH receptors Met and Gce, initiating growth of the midgut that, in turn, increases the digestive capacity of the gut in the mated female . In ente20E can also trigger post-mating ISC proliferation and gut growth . Current+Tdc2, +dsx neuron activity, and increased 20E synthesis in germarium somatic cells) all lie downstream of SP. Thus, signaling pathways downstream of SP can alter signaling both within and between organ systems to coordinate the female PMR.In addition to the coordination afforded by the triggering of relevant PMRs in the digestive and reproductive tracts by the same seminal protein (SP) and its downstream effectors, these organs’ responses are coordinated by signaling crosstalk between the systems. For example, the 20E made in germarium somatic cells stimulates both post-mating ISC proliferation and gut growth ,17. ThusIt is our hope that the network schematic that we provide here can help to organize current knowledge in the field and to provide a framework for making predictions and designing experiments. As new information about the questions we have raised comes to light, or with the identification of entirely new regulators or interactions, the schematic can be revised to update the view of the post-mating signaling events in or between the reproductive and digestive systems.


The role of diet in the pathogenesis of uric acid (UA) nephrolithiasis is incompletely understood. This study investigated the effect of dietary intervention on the risk of UA stone formation under standardized conditions. Twenty patients with idiopathic UA stone disease were included in the study. Dietary intake and 24 h urinary parameters were collected on the usual diet of the patients and a standardized balanced mixed diet. Although urinary UA excretion did not change, the relative supersaturation of UA decreased significantly by 47% under the balanced diet primarily due to the significant increase in urine volume and pH. Urinary pH was below 5.8 in 85% of patients under the usual diet, and in 60% of patients under the balanced diet. The supersaturation of calcium oxalate declined significantly under the balanced diet due to the significant decrease in urinary calcium and oxalate excretion and the increase in urine volume. Dietary intervention is a key component in the management of UA nephrolithiasis. Urinary calcium and oxalate excretion should also be monitored in patients with pure UA calculi to reduce the risk of mixed stone formation with calcium oxalate. Lower urinary pH in UA stone patients can only be partially explained by diet. Uric acid (UA) stones account for about 8% of all urinary calculi ,2,3. UA Characteristic urinary abnormalities in UA stone disease include persistently acidic urinary pH, hyperuricosuria, and low urine volume ,7,8,9. ADue to the high risk of stone recurrence, personalized therapy is of utmost importance in the management of UA stone disease. Dietary factors may affect the urine composition of patients with UA nephrolithiasis. Based on metabolic evaluation and nutritional assessment, dietary treatment should be tailored to the specific metabolic abnormalities and comorbidities of the individual patient. However, information on the dietary patterns of patients with UA calculi is limited. In particular, to our knowledge, studies on the effect of a controlled standardized dietary intervention on the urinary risk profile of UA stone formers are lacking. Therefore, the aim of the present study was to determine urinary abnormalities of UA stone formers under standardized metabolic conditions to exclude exogenous determinants of urinary risk factors for stone formation, especially diet. In addition, we compared urinary composition under the usual, self-selected diet and under the controlled, standardized balanced diet to identify the impact of diet in idiopathic UA stone disease.A total of 20 patients (17 men and 3 women) with UA urolithiasis were included in this study. The patients were admitted to the University Stone Center at the Department of Urology of the University Hospital Bonn for an inpatient metabolic evaluation under standardized conditions. Inclusion criterion was stone analysis of a recent stone episode with at least 50% anhydrous uric acid and/or uric acid dihydrate as stone component. Stone analysis was performed by Fourier transform infrared spectroscopy. Patients did not receive dietary counseling prior to participation in the study and were asked to maintain their usual dietary habits before study entry. Patients did not receive any medication that could affect UA, oxalate and calcium metabolism or acid-base status, such as alkali citrate, allopurinol, or thiazides, for 4 weeks before study entry and during the study. The study was approved by the Ethics Committee of the Medical Faculty of the University of Bonn (430/19) and informed consent was obtained from each patient.Medical history, dietary records, anthropometric, clinical, and 24 h urinary parameters were obtained from all patients at baseline under their usual, self-selected diet. Dietary intake of patients under their habitual diet was recorded using a 7-day food record. Patients provided a detailed description of the types and weighed amounts of all foods consumed. The nutrient composition of the foods was calculated using the PRODI 5.3 computer program . The oxalate content of the foods measured in our laboratory was entered into the database ,22,23. S13C2]oxalate absorption test [13C2]oxalate absorption test was conducted on days 9 and 10 under controlled, standardized conditions.In the following phase, the patients were maintained on a balanced mixed standardized diet for 11 days . After aion test . The [1313C2]oxalate absorption test was performed over a period of two consecutive days under a standardized diet [13C2]oxalate, equivalent to 33.8 mg [13C2]oxalic acid, on the morning of the second day in a fasting state and collected fractional 24 h urines. Unlabeled and labeled oxalate was quantified by gas chromatography–mass spectrometry. Absorption is expressed as a percentage of the labeled oxalate dose. Intestinal oxalate absorption exceeding 10% is defined as hyperabsorption oxalate absorption test was diagnosed with intestinal hyperabsorption greater than 10%. Hypertension was present in 50% and type 2 diabetes in 20% of the patients. Hyperuricemia, defined as serum UA > 6.4 mg/dL, was diagnosed in 40% of patients. Mean serum uric acid concentration was 6.2 ± 1.4 mg/dL on the usual, self-selected diet of UA stone patients and 6.4 ± 1.7 mg/dL on the standardized balanced mixed diet (p = 0.410). A family history of stones was documented in one third of the patients. In 85% of patients, stone analysis revealed 100% UA, with varying proportions of anhydrous uric acid and uric acid dihydrate. The urinary stones of the remaining three patients were admixed with whewellite in proportions of 5%, 15%, and 50%.Twenty patients, 17 men and three women, with idiopathic UA urolithiasis were included in the study. Patient characteristics are shown in The urinary risk profile of UA stone patients on their usual, self-selected diets and the balanced mixed standardized diet is shown in A urinary pH below 5.8 was the most common urinary abnormality on the habitual diet . On the p = 0.410; balanced diet: R = −0.126, p = 0.609) and between urinary pH and BMI  under either diet.No correlation was observed between urinary pH and body weight (usual diet: R = −0.201, Nutrient intake under the usual diet and the balanced mixed diet is depicted in The classic triad of UA nephrolithiasis includes low urine volume, acidic urinary pH, and hyperuricosuria ,7,8,9. SUrinary pH is the major determinant of UA precipitation and stone formation ,9,10. AtSeveral etiologic factors are discussed with regard to acidic urinary pH. A higher dietary acid load from protein consumption has been found to lower urinary pH in healthy subjects ,32. In cThe low urinary pH in UA stone patients has been observed for decades ,35. The 4+/net acid excretion among idiopathic UA stone patients, suggesting that factors other than obesity could contribute to UA stone formation [Several features of the metabolic syndrome, including abdominal obesity, hyperglycemia, and insulin resistance, respectively, have been linked to low urinary pH ,40,41,42ormation . Althougormation . A randoormation .Low urine volume is a primary risk factor for UA stone formation, as the concentration and relative supersaturation of UA increases. In the present study, a urine volume of less than 2.0 L/24 h was diagnosed in 50% of patients on their usual diet. Although urine volume increased significantly with the balanced diet, 35% of the patients still had a urine volume of less than 2.0 L/24 h. It is suggested that the fluid intake with the standardized diet of 2.5 L/d did not compensate for the excessive sweating of the patients. Nutritional counselling should therefore ensure that fluid intake is adapted to individual requirements, which is achieved by the monitoring of urine volume by the patient. In the dietary treatment of UA stone formers, attention should be paid to a generous fluid intake, allowing a 24 h urine volume of at least 2.0 to 2.5 L. Since sufficiently high urine volume and urinary alkalization are essential therapeutic goals in UA urolithiasis, alkalizing beverages, especially bicarbonate-rich mineral waters and citrus juices, should be preferred for recurrence prevention in UA stone disease ,23,45,46Another urinary risk factor that predisposes to UA stone formation is hyperuricosuria. In addition to various genetic and environmental factors, acquired states of UA overproduction, such as a high dietary purine intake, may also contribute to hyperuricosuria . ReducedHyperoxaluria and hypercalciuria are major risk factors for calcium oxalate stone formation. Although 85% of the study population had pure UA stones, hyperoxaluria was present in 25% of patients on the usual diet. Urinary oxalate excretion decreased significantly, and hyperoxaluria could no longer be diagnosed under the balanced diet. The decrease in urinary oxalate excretion was probably due to restriction of dietary oxalate intake. Hypercalciuria was present in 40% of patients on their usual diet but in only 15% of patients on the balanced mixed diet. Several dietary factors are known to affect urinary calcium excretion, particularly the dietary intake of calcium, animal protein, and sodium chloride . Since tThe determination of the urinary risk profile on the usual diet revealed a high risk of UA and calcium oxalate stone formation in UA stone patients. Although urinary UA excretion did not change, the relative supersaturation of UA decreased significantly by 47% under the balanced diet, which was primarily due to the significant increase in urinary pH and volume. Moreover, the relative supersaturation of calcium oxalate declined significantly and to the same extent due to a significant decrease in urinary oxalate and calcium excretion and a significant increase in urine volume under the balanced diet. Estimates of the ion activity products of UA and calcium oxalate confirmed these results. The findings of the present study indicate that urinary calcium and oxalate excretion should also be monitored in patients with pure UA stones to reduce the risk of mixed stone formation with calcium oxalate. Dietary intervention was found to be a key component in the treatment of UA urolithiasis. Due to the alkalizing potential of fruits and vegetables, a lacto-vegetarian diet could be an option to support urinary alkalization. Further studies under standardized conditions are needed to evaluate the effects of a lacto-vegetarian diet on the risk of stone formation in UA nephrolithiasis.To our knowledge, this is the first study to evaluate the effect of dietary intervention under controlled, standardized conditions on the urinary risk profile of UA stone patients. The findings of this study suggest that dietary intervention favorably affects urinary risk factors for UA stone formation, making it an effective treatment strategy for the management of UA nephrolithiasis. Urinary calcium and oxalate excretion should also be monitored in UA patients with pure UA stones to reduce the risk of mixed stone formation with calcium oxalate. Based on the results of this study, a lower urinary pH in UA stone patients can only be partially explained by diet. Although the role of overweight and obesity in UA nephrolithiasis remains unclear, weight reduction is recommended as an additional therapeutic approach to further reduce the risk of UA stone formation.


Deregulated Wnt signaling is responsible for most cases of colorectal cancer (CRC). Dietary fiber is protective against CRC and this activity is likely mediated by butyrate, a breakdown product of dietary fiber that hyperactivates Wnt signaling, repressing CRC proliferation and inducing apoptosis. Receptor-mediated Wnt signaling and oncogenic Wnt signaling, which is typically initiated by mutation in more downstream elements of the pathway, activate non-overlapping patterns of gene expression. Receptor-mediated signaling is associated with a poor prognosis for CRC while oncogenic signaling is associated with a relatively good prognosis. We have compared the expression of genes differentially expressed in receptor-mediated vs. oncogenic Wnt signaling to microarray data generated in our laboratory. Most importantly we evaluated these gene expression patterns comparing the early stage colon microadenoma line LT97 with the metastatic CRC cell line SW620. LT97 cells exhibit a gene expression pattern more strongly associated with that observed with oncogenic Wnt signaling, while SW620 cells exhibit a gene expression pattern moderately associated with that observed with receptor-mediated Wnt signaling. Given that SW620 cells are more advanced and malignant compared to LT97 cells, these findings are generally consistent with the better prognosis observed with tumors exhibiting a more oncogenic Wnt gene expression pattern. Importantly, LT97 cells are more sensitive to the effects of butyrate on proliferation and apoptosis that are CRC cells. We further examine these gene expression patterns in butyrate-resistant vs. butyrate-sensitive CRC cells. Based upon all of these observations, we hypothesize that colonic neoplastic cells exhibiting a more oncogenic as compared to receptor-mediated Wnt signaling gene expression pattern would be more sensitive to the effects of butyrate, and, hence, fiber, than are those cells exhibiting a more receptor-mediated Wnt signaling pattern of expression. Diet-derived butyrate may affect the differential patient outcomes resulting from the two types of Wnt signaling. We further posit that development of butyrate resistance and concomitant changes in Wnt signaling patterns, including associations with CBP and p300, disrupts the association between the two major types of Wnt signaling (receptor-mediated and oncogenic) and neoplastic progression/prognosis. Ideas about testing the hypothesis and therapeutic implications are briefly considered. Deregulated Wnt signaling is responsible for most cases of colorectal cancer (CRC) vice versaThe histone acetylases CREB Binding Protein (CBP) and p300 activate different sets of Wnt target genes and therefore have different phenotypic consequences for cells APC KO) Wnt signaling is markedly different from that induced by receptor-mediated signaling Using a human colon organoid approach, it was shown that the gene expression profile resulting from oncogenic  of butyrate.ADAMTS14 and SLC2A3 are more highly expressed in SW620 cells than in LT97 cells (+/- butyrate); however, SMOC1 unexpectedly exhibits the opposite pattern CKB, FCGBP, ID3, MTMR11, and VAV3 are all more highly expressed in early-stage LT97 adenoma cells (+/- butyrate) compared to metastatic SW620 cells, while ATOH8 is more highly expressed in LT97 cells but only in the absence of butyrate ATOH8. It should be noted that both SW620 and LT97 cells exhibit oncogenic Wnt signaling due to APC mutation, similar to the colon organoids of the Michels et al. study Most important is the comparison of gene expression between SW620 metastatic CRC cells and the early-stage microadenoma line LT97 ADAMTS14 is downregulated in HCT-R cells compared to HCT-116 cells, while SLC2A3 shows the opposite pattern (both +/- butyrate). ATOH8 is upregulated and FCGBP is downregulated in HCT-R cells compared to HCT-116 cells (both +/- butyrate), while CKB is downregulated in HCT-R cells in the presence of butyrate, compared to HCT-116 cells. HCT-R cells exhibit downregulates expression of p300, which can be a cofactor in Wnt signaling, and we have shown that this downregulation is associated with butyrate resistance ADAMTS14 is downregulated in the KO cells, as is expression of ASIC1, while MTMR11 is modestly upregulated . One reason why the data for the butyrate resistant cell line vs. HCT-116 comparisons are not as clear-cut as that of SW620 vs. LT97 is that while SW620 cells are clearly more advanced along the neoplastic spectrum than are LT97, the situation for the butyrate-resistant lines are not as clear. On the one hand, loss of p300 can be associated with worse prognosis ; p300 KO lines show increased migration in culture in vivo tumorigenic potential of HCT-R vs. HCT-116 cells has not yet been evaluated. It is possible that butyrate-resistant cells, which have disrupted response of Wnt signaling to butyrate, with decreased p300 responses, exhibit a disrupted association between the two major types of Wnt signaling (receptor-mediated and oncogenic) and neoplastic progression/prognosis  would constitute part of the approach to test the hypothesis (below).The researchers Detailed clinical, cellular, biochemical, and molecular comparisons between oncogenic and receptor-mediated Wnt signaling, with respect to the issues covered in this manuscript, are summarized in Table per se, but also to a particular molecular signature that is naturally less sensitive to the effects of butyrate. Given that CMS4 tumors tend to have a worse prognosis, a high fiber diet would have the advantage of reducing overall risk of CRC, but when CRC does develop, it may have a worse prognosis. We further posit that development of butyrate resistance and concomitant changes in Wnt signaling patterns, including associations with CBP and p300, disrupts the association between the two major types of Wnt signaling (receptor-mediated and oncogenic) and neoplastic progression/prognosis.We hypothesize that colonic neoplastic cells exhibiting a more oncogenic as compared to receptor-mediated Wnt signaling gene expression pattern would be more sensitive to the effects of butyrate, and, hence, fiber, than are those cells exhibiting a more receptor-mediated Wnt signaling pattern of expression. This main hypothesis allows us to derive further, consequent hypotheses. Diet-derived butyrate may affect the differential patient outcomes resulting from the two types of Wnt signaling. Thus, levels of butyrate in the colonic lumen would hyperactivate Wnt signaling and induce apoptosis to a greater degree in patients with tumors exhibiting more of an oncogenic Wnt signaling gene expression pattern than those with tumors of the receptor-mediated type. These differences could decrease tumor load, and affect tumor grade, probability of metastasis, as well as response to treatment. It is possible that differences in the types of tumors  that are found in patients are affected by dietary history. Thus, a high fiber diet, with higher levels of colonic butyrate, may select against the development of CMS2 tumors  as opposed to CMS4 tumors . Thus, development of CRC in the context of a high fiber diet may not only be due to butyrate resistance The hypothesis can be tested by three approaches Fig. . First, CBP or p300 knockout  can also be utilized, although one would expect a greater number of (possibly off-target) effects by a general knockout of these factors, as opposed to a more targeted pathway inhibition with the pharmacological agents. However, use of Wnt inhibitors can be used to better define the Wnt signaling-dependent components of the effects of CBP or p300 knockout, with ICG-001 more consistently mimicking CBP ablation in vivo; however, first genetic engineering of CRC cells should be performed to ascertain what components of the receptor-mediated vs. oncogenic Wnt signaling molecular signatures are required for effects of butyrate treatment and to optimize response or resistance to the effects of butyrate treatment. Thus, CRC cells can be modified to reflect gene expression patterns similar to that found in receptor-mediated Wnt signaling organoids, oncogenic Wnt signaling organoids, or in butyrate-resistant cells. Cells can be stably transfected with expression vectors for the relevant genes, and CRISPR can be used to knockout other relevant genes, depending on whether increased or decreased expression is desired. The gene expression patterns used to model these engineered cells would be derived from ref. 13, as well as from microarray data from our laboratory Next, the hypothesis can be evaluated The resulting cells could then be tested according to the same metrics outlined above and an iterative approach used to redesign the cells to reflect the optimized phenotypes expected for each cell type. This approach could be used to evaluate the extent that CBP-Wnt vs. p300-Wnt signaling is involved in the differential outcomes of receptor-mediated Wnt signaling vs. oncogenic Wnt signaling, particularly in conjunction with butyrate treatment. CBP-Wnt signaling would be expected to be relatively more important in neoplasia that is derived more from receptor-mediated Wnt signaling, while the oncogenic Wnt signaling profile would be expected to exhibit a relatively greater reliance on p300 and p300-Wnt activity. Knockout and/or overexpression of these two factors may be an important component of the iterative process of engineering cells to express the optimized molecular signatures and cell phenotypes characteristic of the organoid types studied in ref. 13.The optimized cells could then be implanted in nude mouse models, and tumorigenesis and survival assayed under different dietary conditions or with treatment with butyrate. One would expect that cells with an “extreme” receptor-mediated Wnt molecular signature would be butyrate resistant, similar to cells designed to be butyrate-resistant , and would be more aggressively tumorigenic in animal models. Conversely, cells with optimized oncogenic Wnt molecular profiles would be more sensitive to butyrate, and less tumorigenic in mouse models. Dietary interventions leading to increased levels of colonic butyrate would be more effective in orthotopic nude mouse models in which oncogenic Wnt signaling-optimized tumors are implanted into the colon, as opposed to receptor mediated Wnt signaling-optimized tumors.Finally, the relationship between patient dietary history and tumor type can be ascertained Fig. C. AccordWhile dietary fiber is protective against CRC, likely in part due to the activity of butyrate, resistance to butyrate would decrease the preventive efficacy of a high fiber diet The degree of Wnt hyperactivation exhibited by the different Wnt signaling pathways may be mediated by effects of CBP and p300 on Wnt activity and consequent gene expression. Therefore, we expect that different classes of CRC, representing greater or lesser degrees of receptor-mediated vs. oncogenic Wnt signaling will exhibit differential sensitivity to butyrate and to therapeutic HDACis, with clinical significance. This may be amenable to modulation by specific inhibitors of CBP-Wnt vs. p300-Wnt activity Finally, we note the relevance and novel aspects of this work compared to the general field of butyrate studies on CRC, as well as our own work in this field. While there has been much focus on the role of butyrate in mediating the preventive effects of fiber against CRC, our work on this topic has been unique in relating these effects of butyrate to the fundamentally important Wnt signaling pathway. The first observation that the Wnt signaling pathway can be pharmacologically modulated, particularly by histone deacetylase inhibitors (including butyrate), and the physiological implications of this for CRC prevention, was from the work of the present author, in the Augenlicht laboratory


Here, we demonstrate using various fluorescence imaging approaches at the single cell level to measure kinetics of (i) receptor activation, (ii) receptor signaling via Gs and Gq, and (iii) receptor internalization and recycling that the native PTHrP1-141 displays biased agonist signaling properties that are not mimicked by PTHrP1-36. Although PTHrP1–36 induces transient cAMP production, acute intracellular Ca2+ (iCa2+) release and β-arrestin recruitment mediated by ligand–PTHR interactions at the plasma membrane, PTHrP1-141 triggers sustained cAMP signaling from the plasma membrane and fails to stimulate iCa2+ release and recruit β-arrestin. Furthermore, we show that the molecular basis for biased signaling differences between PTHrP1-36 and properties of native PTHrP1-141 are caused by the stabilization of a singular PTHR conformation and PTHrP1-141 sensitivity to heparin, a sulfated glycosaminoglycan. Taken together, our results contribute to a better understanding of the biased signaling process of a native protein hormone acting in conjunction with a GPCR.The parathyroid hormone (PTH)–related protein (PTHrP) is indispensable for the development of mammary glands, placental calcium ion transport, tooth eruption, bone formation and bone remodeling, and causes hypercalcemia in patients with malignancy. Although mature forms of PTHrP in the body consist of splice variants of 139, 141, and 173 amino acids, our current understanding on how endogenous PTHrP transduces signals through its cognate G-protein coupled receptor (GPCR), the PTH type 1 receptor (PTHR), is largely derived from studies done with its N-terminal fragment, PTHrP Developments in recording GPCR-signaling cascade in individual cells in real time using optical approaches during the decade of the ‘00s , cytosolic adapter proteins that canonically act to occlude further G protein coupling and promote translocation of the ligand–receptor complex from the cell surface to early endosomes. In contrast, PTH1-34 causes an additional sustained phase of cAMP generation via PTH–PTHR–βarr complexes that remain active in early endosomes. Thus, this distinction in the spatiotemporal cAMP profiles of PTH and PTHrP was proposed to be the underlying determinant responsible for their biological specificity.Upon its activation, the parathyroid hormone (PTH) receptor (PTHR) triggers both Gthe ‘00s , 2 have 1-34 and PTHrP1-36, retain their full capacity to stimulate adenylyl cyclase in cAMP accumulation assays led to the utilization of these N-terminal fragments in most studies. Indeed, it was PTH1-34 and PTHrP1-36 that were used in the aforementioned work that revealed differences in the time courses and subcellular locations of cAMP production by these two peptides. In contrast to these earlier findings of transient signaling by PTHrP1-36, a recent publication proposed sustained endosomal cAMP generation induced by full-length PTHrP1-141  and without a significant difference in the hormone concentration dependence (C and D). We observed a striking inability of PTHrP1-141 to efficiently induce the release of intracellular calcium (iCa2+) from the endoplasmic reticulum (E and F), indicating defective Gq activation by PTHrP1-141. We have previously shown that Gq activation is required for endosomal cAMP generation by PTH1-34  or uncoupled (R0) states of PTHR (G and H) but were rather due to the stabilization of a distinct receptor conformation. We tested this theory by using cells expressing a FRET-based PTHR sensor . Time-resolved determination of intramolecular FRET changes recorded from single cells allows the analysis of the kinetics of receptor activation in response to ligand binding  for receptor activation indicated the stabilization of distinct PTHR conformations (I and J).We utilized FRET to record real-time courses of cAMP production in single HEK293 cell stably expressing PTHR (HEK-PTHR). We found that brief stimulation with PTHrPTHrP1-36 A and S1. similar B and witpendence , C and Deticulum , E and F of PTHR , G and H binding . As expevia FRET in cells transiently expressing PTHRCFP and βarr-2YFP. The βarr2 isoform was randomly selected, given that earlier studies demonstrated that PTH1-34 and PTHrP1-36 displayed equal potencies  for recruitment of both β-arr1 and β-arr2 (1-34 resulted in significant association of βarr with the receptor that was stably maintained following ligand washout (A). In contrast, analogous experiments using PTHrP1-141 failed to promote this interaction (A), suggesting that the sustained signaling observed for PTHrP1-141 occurs in a βarr-independent manner. This finding led us to test the role of receptor internalization, a key step in PTHR endosomal signaling. Measurements of receptor internalization and recycling in single cells stably expressing PTHRSEP, the PTHR N-terminally tagged with a pH-sensitive GFP (super-ecliptic pHluorin SEP) that exhibits fluorescence intensity reduction in the acidic environment encountered in endosomes , showed reduced internalization and faster recycling in response to PTHrP1-141 or PTHrP1-36 when compared to PTH1-34  . Strikingly, blockade of receptor internalization significantly reduced the magnitude and duration of cAMP production by PTH1-34 (C and E) but had no effect on cAMP mediated by PTHrP1-141 (D and E), indicating that native PTHrP does not promote sustained signaling in an endocytosis-dependent manner. We recently reported on the development of Gs-biased PTH analogs that stimulate sustained cAMP production exclusively from the cell surface due to retention of active ligand–receptor complexes at the cell surface. This was experimentally confirmed via cAMP time courses using a cell-impermeable PTHR antagonist, which completely abolished the sustained phase of cAMP generation for GS-biased peptides but not for PTH1-34, consistent with its ability to signal from intracellular compartments . These findings demonstrate that PTHrP1-141 promotes sustained cAMP responses via active ligand–receptor complexes localized to the cell surface, which appear inconsistent with experiments showing receptor internalization.To assess the role of βarr recruitment, we measured PTHR–βarr interactions d β-arr2 , 11, 12. washout A. In coneraction A, sugges PTH1-34 B and S2.response . Accordiartments . We thus1-141 (88KKKKGKPGKRKEQEKKKRRTR108), not present in PTHrP1-36 or PTH, permits the hormone to attach to the cell surface via interactions with polyanionic glycosaminoglycans (GAGs) present on membrane glycoproteins such as heparan sulfate proteoglycan. Consistent with this theory was the significant reduction in the magnitude and duration of cAMP production in response to PTHrP1-141 in the presence of soluble heparin used as a decoy to prevent potential PTHrP1-141 and GAGs interactions (A and B). The selective effect of heparin was verified by its lack of inhibitory action on cAMP induced by either PTH1-34 or PTHrP1-36 .Collectively, these data prompt a reinterpretation of our previous understanding on how hormones act on the PTHR by providing compelling evidence that native PTHrP hormone C.Materials and methods are detailed in SI Appendix.http://d-scholarship.pitt.edu/).Source data are stored in Excel 2013 and will be deposited in the institutional repository of the University of Pittsburgh (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Skeletal muscle mass is determined by the balance between muscle protein synthesis (MPS) and degradation. Several intracellular signaling pathways control this balance, including mammalian/mechanistic target of rapamycin (mTOR) complex 1 (C1). Activation of this pathway in skeletal muscle is controlled, in part, by nutrition  and exercise ). Acute and chronic alcohol use can result in myopathy, and evidence points to altered mTORC1 signaling as a contributing factor. Moreover, individuals who regularly perform RE or vigorous aerobic exercise are more likely to use alcohol frequently and in larger quantities. Therefore, alcohol may antagonize beneficial exercise-induced increases in mTORC1 pathway signaling. The purpose of this review is to synthesize up-to-date evidence regarding mTORC1 pathway signaling and the independent and combined effects of acute alcohol and RE on activation of the mTORC1 pathway. Overall, acute alcohol impairs and RE activates mTORC1 pathway signaling; however, effects vary by model, sex, feeding, training status, quantity, etc., such that anabolic stimuli may partially rescue the alcohol-mediated pathway inhibition. Likewise, the impact of alcohol on RE-induced mTORC1 pathway signaling appears dependent on several factors including nutrition and sex, although many questions remain unanswered. Accordingly, we identify gaps in the literature that remain to be elucidated to fully understand the independent and combined impacts of alcohol and RE on mTORC1 pathway signaling. When the rate of MPS exceeds the rate of MPB, the net result is protein accretion (hypertrophy). A well-known, efficacious method of increasing muscle size is performance of repeated bouts of resistance exercise (RE). RE increases overall protein turnover by concomitantly increasing the rates of both MPS and MPB. Following a single bout of RE, the rate of MPS is increased above rest in the early recovery period e.g., 1–5 h) [–5 h 1,22,3 throulalanine ,5 and MPlalanine ,5. This 35S-methionine/cysteine incorporation, but no change in MPB measured using 35S-methionine/cysteine release, was also observed when human myocytes were cultured with ethanol or its major metabolites, acetaldehyde and acetate, for 24–72 h [Physically active individuals, especially those who regularly perform RE and vigorous-intensity exercise, report consuming alcohol more frequently and in larger quantities compared to sedentary individuals ,7,8,9. U 24–72 h . Further 24–72 h , with no 24–72 h . TogetheProtein synthesis is controlled, at least in part, by highly conserved cell signaling pathways. Key among these is the mechanistic/mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. A variety of stimuli, including ethanol and RE, can modulate the activity of this pathway and thus contribute to increases or decreases in MPS. For example, administration of rapamycin, a potent inhibitor of mTORC1, to human subjects prevented the RE-induced increase in MPS observed in the early (1–2 h) post-exercise period, at least in part by blocking, attenuating, or delaying key events within the mTORC1 pathway . AdminisImportantly, muscle hypertrophy results from repeated bouts of RE  that allow for a net protein accretion over time, thus the anabolic stimulus induced by each acute bout is important. Ethanol and RE can independently alter the mTORC1 pathway and MPS. When consumed before or after a bout of RE, ethanol can attenuate RE-induced mTORC1 pathway signaling and, therefore, ethanol ingestion might be detrimental to maximizing RE-induced MPS ,18,19,20The mTOR protein exists in two distinct multiprotein complexes. In complex 1 (mTORC1), mTOR is associated with the regulatory associated protein of mTOR (Raptor), proline-rich Akt substrate of 40-kDa (PRAS40), mammalian lethal with sec-13 , and DEP-domain-containing mTOR-interacting protein (Deptor). In complex 2 (mTORC2), mTOR is associated with the raptor-independent companion of mTOR (Rictor), mLST8, Deptor, protein observed with Rictor (Protor), and mammalian stress-activated protein kinase-interacting protein (mSin1). Each complex has unique upstream regulation and downstream targets. Rapamycin inhibits signaling through mTORC1 by binding to its intracellular receptor, FK506-binding protein 12 (FKBP12), and the subsequent FKBP12-rapamycin complex binds to the FKBP12-rapamycin binding (FRB) domain of mTOR . This biThe mTOR signaling pathway is a nutrient, growth factor, and mechanically sensitive anabolic pathway that plays a key role in load-induced SKM hypertrophy. Different stimuli change the activation state of a variety of upstream components of the pathway to regulate the kinase activity of mTOR. Once activated, mTOR exerts its downstream effects. It should be noted that other pathways, such as the MAPK pathways, also promote protein synthesis and converge with the mTOR signaling pathway at several locations. Major outcomes of the activation of the mTOR signaling pathway include translation initiation, peptide elongation, ribosomal biogenesis, suppression of autophagy, and ultimately, protein synthesis .In canonical mTOR pathway signaling, the ligands insulin and insulin-like growth factor-1 (IGF-1) bind and activate their membrane-bound receptors, resulting in signal transduction to phosphoinositide-3-kinase (PI3K) ,35. PI3KTSC2 resides within a complex alongside tuberous sclerosis complex 1  and TBC1D7 . TSC2 isThe specific upstream mechanism by which a mechanical stimulus  transduces a signal to enable mTOR activation is poorly understood but attempts to elucidate this mechanism have provided substantial insight. The activation of mTOR in response to mechanical stimuli bypasses signaling through PI3K/Akt ,46. InstThe generation of phosphatidic acid (PA) also contributes to mechanically-induced stimulation of mTOR signaling . AlthougT389 phosphorylation . While the GTP-loading state of RagA/B mediates amino acid-dependent mTOR activation [Nutrient availability affects signal transduction through the mTOR signaling pathway. Amino acids, particularly (but not solely) leucine, potently stimulate signaling through mTOR ,63, althtivation ,65,66, htivation , but Ragtivation ,70. Undetivation . Under ativation , inhibittivation , and thutivation ,74,75.Alternatively, signal transduction through mTOR is repressed under conditions of nutrient deficiency  or cellular stress . Under these cellular conditions, AMP-activated protein kinase (AMPK) is activated by phosphorylation on Thr172  and, in Ser422 [S422 represents a point of convergence between the mTOR and MAPK signaling pathways [Upon activation, mTOR phosphorylates its downstream substrates including ribosomal protein S6 kinase-1  , an impoSer422 , indicatpathways  and thatpathways . MoreoveS366 [S235/236 [Beyond its role in translation initiation, S6K1 also indirectly regulates peptide chain elongation and ribosomal biogenesis. S6K1 phosphorylates and inhibits eukaryotic elongation factor 2 kinase  , which rS366  and rpS6S235/236 .Overall, mTOR integrates signals from growth factors, nutrients, and mechanical stimuli. In stressful or nutrient-poor conditions, mTOR kinase is inactive. In nutrient-rich and anabolic environments, mTOR can phosphorylate its downstream substrates to promote translation. Two factors that can acutely alter the activation of various components of the mTOR signaling pathway are ethanol and RE. Current knowledge regarding the effects of each alone and the combined effects of ethanol and RE on mTOR pathway signaling will be discussed in subsequent sections.Although the exact mechanisms by which ethanol adversely affects MPS have not been fully established, a substantial amount of evidence exists to demonstrate that acute ethanol administration alters the activation state of a variety of key proteins within the mTOR signaling pathway. Furthermore, ethanol can alter the anabolic responses to stimuli such as leucine, insulin, and IGF-1 in such models. This portion of the review will focus on describing the ethanol-induced changes to mTOR pathway signaling in the basal state and in response to amino acids or hormone administration. Much of the literature relies on preclinical or cell culture-based models. Rodents have higher rates of ethanol clearance than humans ,100, so T172 compared to control cells [S1387 [T172 and TSC2S1387, indicating that it is possible for anabolic agents to “balance” the anti-anabolic effects of ethanol. It is important to note that ethanol can also interfere with the endogenous production of PA by preferentially converting phosphatidylcholine to phosphatidylethanol  consistently display increased phosphorylation of AMPKol cells ,108,109 s [S1387 ,109. Ints [S1387  or PA [1iewed in ), potentrylation ,112,113 rylation ,112 in trylation . Given tS792 and mTOR/Raptor association [AMPK can also suppress mTOR activity by phosphorylating the mTOR-associated protein, Raptor (Ser722 and Ser792), independently of TSC2 [ociation . Furtherociation . The relociation , indicatociation . It is pEthanol also affects the ability of amino acids to stimulate signaling through mTOR ,118. ThiT172 and TSC2S1387, and these ethanol-induced changes were prevented by co-incubation with leucine [S2448 and AMPKT172 in murine myotubes and SKM from patients with alcohol-related liver disease [The Rag-Ragulator complex aids in mTOR signal transduction, at least in part, by tethering the mTOR/Raptor complex to the lysosome, where Rheb-GTP can bind and activate mTOR. Incubating C2C12 myocytes with ethanol reduced the association between mTOR and Rheb . Althoug leucine . The pro disease . AlthougT308 phosphorylation, despite ethanol-induced changes in hormone-stimulated phosphorylation of proteins downstream of mTOR [T308 and AktS473 compared to control animals despite greater circulating insulin concentrations 1 h after ethanol administration [In addition to its effects on the AMPK/TSC2/Rheb and Rag-Ragulator branches upstream of mTOR, evidence exists, albeit mixed, regarding the effects of acute ethanol on components of the PI3K/Akt portion of the pathway upstream of mTOR. In male rats, acute ethanol (75 mmol/kg body mass) did not affect the basal levels or insulin-induced increase in insulin receptor and insulin receptor substrate (IRS)-1 phosphorylation . IGF-1 i of mTOR . In contstration , indicatstration . TherefoS2448 phosphorylation is consistently detected in C2C12 myocytes incubated with ethanol (100 mM) for 18–24 h [S2448 phosphorylation. Basal phosphorylation of 4E-BP1T37/46 is also reduced with ethanol in C2C12 myocytes [T37/46 phosphorylation was not affected by ethanol 1 h after administration in female mice despite a BAC of approximately 0.27 g/dL and an overall reduction in MPS [In addition to the upstream alterations in mTOR pathway signaling observed with ethanol, changes downstream of mTOR have also been observed, but not all components downstream of mTOR are similarly and simultaneously affected. Reduced mTOR 18–24 h ,115,123, 18–24 h , and in  18–24 h . In addi 18–24 h , which imyocytes ,109,123 myocytes ,118,124.myocytes ,118,123,n in MPS , indicatT389 phosphorylation in response to incubation with 100 mM ethanol for 18–24 h is consistently observed [S235/236 phosphorylation [S366 phosphorylation [T56 phosphorylation [T389 phosphorylation after 1–2.5 h [S235/236 and rpS6S240/244 in vivo [T56 in the psoas muscle [In contrast to the consistent findings across models for the inhibitory effects of ethanol on mTOR, findings for the effect of ethanol on basal phosphorylation of S6K1, a direct downstream target of mTOR, appear to differ between models. In C2C12 myocytes, reduced S6K1observed ,115,123 rylation ,123, redrylation , and incrylation ,108,123. 1–2.5 h ,122,125. in vivo ,122,125.s muscle , which is muscle . Althougs muscle . MoreoveS2448, 4E-BP1T37/46, S6K1T389, and rpS6S235/236 [T70 and γ isoform but not 4E-BP1T37/46, S6K1T389 and S6K1T421/S424, and rpS6 (site not indicated), and attenuated the leucine-induced increase in phosphorylation of mTORS2448 [T389 and rpS6S235/236 phosphorylation 2.5 h after ethanol administration regardless of route of administration  but did not alter the IGF-1-induced increase in 4E-BP1T37/46 phosphorylation or decrease in 4E-BP1 bound to eIF4E. Furthermore, a single dose of ethanol (75 mmol/kg body mass) prevented the IGF-1 induced increases in S6K1T389 and rpS6S235/236 phosphorylation 1 and 4 h after administration, and these decreases persisted through 8 h after administration [T37/46 phosphorylation or decrease in 4E-BP1 bound to eIF4E at 1, 4, 8, or 24 h after administration [T389 phosphorylation. Ethanol also attenuated the insulin-, IGF-1, and refeeding-induced increases in rpS6 phosphorylation [Ethanol can also alter changes in signaling downstream of mTOR in response to the administration of anabolic agents such as leucine, insulin, and IGF-1. For example, co-incubation of C2C12 myocytes with leucine and ethanol in vitro prevented the leucine-induced increases in phosphorylation of mTORS235/236 . In vivoTORS2448 . EthanolTORS2448 . HoweverTORS2448 , which cTORS2448 . AdditioTORS2448  found thstration . The samstration . In a sestration  also fourylation ,124. Howrylation ,124. Ethrylation , a findirylation  regardinrylation  observedA landmark study by Baar and Esser in 1999  demonstrT172 phosphorylation can persist at 1 h after exercise in the fasted state [S9 phosphorylation, which inhibits mTOR signaling by phosphorylating and activating TSC2 [T308 and AktS473 has also been observed immediately after RE [T1462 (an Akt target) [S2448 and S6K1T389 phosphorylation appear unchanged immediately to 10 min after RE in the fasted state [T37/46 phosphorylation immediately to 10 min after RE has been reported [Immediately after an acute bout of RE, the phosphorylation of upstream components of the mTOR signaling pathway largely indicate inhibitory input toward mTOR in human skeletal muscle. For example, AMPK activity  and phosed state ,132. Thied state , which ied state . Furthering TSC2 , was incing TSC2 . Decreasafter RE ; howeverafter RE  or TSC2Ted state ,140. Howed state ,142, inded state . Decreasreported ,140, whireported , suggestS2448 [T389 [S235/236 and rpS6S240/244 [T56 [In contrast to immediately after exercise, mTOR pathway activation is upregulated later into the RE recovery period . For example, in untrained, fasted individuals, mTORS2448 ,144,145,48 [T389 ,144,145,S240/244 ,141,143 244 [T56 ,141,144 244 [T56 ,132,144.Although conducting investigations in which participants remain fasted allows for elucidation of the specific effects of RE on intramuscular signaling and MPS without the influence amino acid- or insulin-induced signaling, this design is likely less externally valid compared to a design which incorporates feeding before and/or after the exercise bout. Furthermore, RE and feeding each stimulate mTOR pathway signaling and MPS, and the combined effects are greater than either stimulus alone ,147,148.S2448 and S6K1T389 phosphorylation at 1, 3, and 6 h and similar decreases in eEF2T56 phosphorylation at 3 and 6 h into recovery were observed, but the increased 4E-BP1T37/46 phosphorylation observed in both groups at 3 h persisted to 6 h after RE only for the younger men [Age also affects anabolic responses to an acute bout of RE in the fed  and fastnger men . Since 4nger men . It woulnger men . Attenuanger men  suggestinger men . Dose-denger men . For oldnger men . Sex difnger men .S240/244 phosphorylation 5 h after exercise, and increases the duration of enhanced sensitivity to amino acids  [S473 and mTORS2448 phosphorylation, respectively, one day following RE, whereas performance of a work-matched leg extension protocol using 30% of 1 RM  did not result in such increases [S2448 and AktS473 phosphorylation 5 h after RE, possibly due to the combination of strenuous RE and low nutrient intake provided to participants [Acute program variables such as volume and intensity of exercise are also important factors in the magnitude of the anabolic response to RE. Performance of 3 sets versus 1 set of lower body resistance exercise elicits a more substantial increase in myofibrillar MPS, increases rpS6ing day) . Similarncreases . Therefoncreases . Interesicipants . TherefoT389 and rpS6S235/236 phosphorylation increased to a greater extent than in those who had undergone 12 or 18 bouts of stimulated muscle contractions  [T389 phosphorylation immediately and 4 h after exercise in untrained SKM, but only immediately after exercise in trained SKM using the same relative workload [S235/236 phosphorylation was increased 4 h after exercise in untrained muscle, but this increase was not observed after training regardless of whether a constant or progressive loading scheme was used [T421/S424 and rpS6S235/236 phosphorylation similar to that of the first RE session [Training status also influences mTOR pathway signaling and MPS in response to RE. Only a few investigations have directly compared acute responses to RE in trained and untrained SKM, but several of these studies have demonstrated a blunted anabolic response in trained muscle. For example, when rats performed an acute bout of stimulated muscle contractions (designed to mimic RE) in an untrained state, S6K1c force) , indicatworkload . Similarwas used . However session . Strengt session .The protein synthetic response also appears to become more targeted after RE training. For example, when untrained individuals performed an acute bout of RE, MPS increased for the myofibrillar and the mitochondrial fractions, but after training, only an increase in myofibrillar MPS was observed . FurtherOverall, acute RE appears to result in decreased mTOR pathway signaling during and immediately after exercise with a concomitant decrease in MPS followed by increased mTOR pathway signaling during the 6 h after exercise. Moreover, given sufficient volume, an acute bout of RE appears to increase the anabolic responsiveness of muscle to amino acids at least through 29 h after exercise. Factors such as nutrition, age, acute program variables, and training status impact the magnitude of mTOR pathway signaling responses, rates of MPS, and the duration of the elevation in MPS.As described above, acute ethanol and RE independently impact mTOR pathway signaling, but in opposite directions such that acute ethanol generally has suppressive effects, whereas RE generally has stimulatory effects. In contrast to the independent effects of ethanol and RE on mTOR pathway signaling, few studies have investigated the combined effects of ethanol and RE on mTOR pathway signaling and MPS. Recreational ethanol use is higher among physically active individuals compared to sedentary individuals ,7,8, espT389, 4E-BP1 (γ isoform), and rpS6S240/244 phosphorylation 30 min after muscle contraction [T389, rpS6S240/244, 4E-BP1 (γ isoform), and mTORS2448 were absent in the rodents who received alcohol prior to contraction [T389 and rpS6S240/244 were still prevented or attenuated, respectively, in rats who had been administered ethanol [T389, rpS6S235/236 and rpS6S240/244, or 4E-BP1T37/46 phosphorylation 4 h after contractions [T56 phosphorylation [The combined effects of ethanol and RE on mTOR signaling have been investigated in rodents and in humans. In the first rodent study examining this question, ethanol (3 g/kg) was administered prior to unilateral electrically stimulated muscle contractions and resulted in attenuation or prevention of contraction-induced S6K1traction . By 4 h traction . By 12 h ethanol . Similar ethanol . By 12 h ethanol . Thus, eractions . Howeverrylation . Despiterylation , possiblS2448 phosphorylation 2 h, but not 8 h, after exercise and the rate of myofibrillar MPS measured between 2 and 8 h after exercise compared to post-exercise protein ingestion, but did not affect S6K1T389 or 4E-BP1T37/46 phosphorylation [T56 phosphorylation was reduced at 2 and at 8 h after exercise only when ethanol was consumed post-exercise [S2448 and S6K1T389 phosphorylation 3 h after exercise in men but not in women [S2448, S6K1T389, rpS6S235/236, and eEF2KS366 phosphorylation and decreased eEF2T56 phosphorylation 2 h after RE; however, ethanol did not affect these results (unpublished observations). One major difference between the two studies was nutrient timing. In the first, a standardized meal was administered 65 min prior to exercise (4 h before the first post-exercise biopsy) [Several investigations of the effects of ethanol on exercise-stimulated mTOR pathway signaling and protein synthesis have been carried out in humans. For example, ethanol  co-ingested with protein after a bout of concurrent RE and aerobic exercise attenuated the exercise-induced increase in mTORrylation . Interesexercise , which iexercise  and fracexercise  responseexercise . Consumiin women . These e biopsy) , so it i biopsy) ,150,173.There is substantial evidence regarding independent and opposite acute effects of ethanol and RE on mTOR pathway signaling, and some evidence supporting acute ethanol attenuating the RE-induced increase in activation of this pathway. Therefore, there is reason to believe that regularly consuming ethanol during the post-RE period could negatively impact hypertrophic adaptations. To date, no study has evaluated this question in humans using traditional RE training, and few studies have examined this or similar questions in preclinical models. The single study directly examining whether chronic ethanol impacts overload-induced muscle growth found that ethanol consumption described as moderate (~20 g/kg/day for rodents) did not attenuate overload-induced hypertrophy  in male mice . Others Only the BEER-HIIT study has evaluated the impacts of regular ethanol consumption on exercise induced adaptations in humans ,178, andAlterations in SKM size are determined by the relative rates of MPS and MPB over time. The mTOR signaling pathway has been identified as an important mediator of the translational process, is critical for SKM hypertrophy, and is controlled by hormones, nutrients, and mechanical stimuli. However, the specific mechanisms by which amino acids and mechanical stimuli activate the mTOR pathway remain to be fully elucidated. Acute ethanol ingestion suppresses MPS and components of the mTOR signaling pathway, and the exact mechanism for this suppression is also not yet established. Notably missing from the literature are dose-response studies examining the impact of different amounts of ethanol on mTOR pathway signaling. Whether differences exist based on the type of ethanol-containing beverage consumed  should also be explored since other beverage components may have confounding impacts on mTOR signaling and MPS. In contrast to ethanol, acute RE increases the rates of MPS and MPB, but given sufficient nutrients, net protein balance is positive after RE. Acute RE also increases mTOR pathway signaling. Few investigations have explored the combined effects of ethanol and RE on mTOR pathway signaling and MPS. Overall, it appears that ethanol can, but does not always, suppress RE-induced increases in mTOR pathway signaling and protein synthesis, and this suppression is likely dependent on the specific exercise protocol, timing of ethanol intake, dose of ethanol, nutrient co-ingestion, and sex of the individuals. Since increased mTOR pathway signaling can persist long after a bout of RE, and ethanol may not be consumed in the early post-RE period, the impact of ethanol consumed later in the recovery period  on mTOR pathway signaling in skeletal muscle would be highly relevant. The effects of ethanol on RE-induced anabolic signaling should be further explored since RE is a potent stimulus for increasing muscle size and could be a useful method of counteracting and preventing muscle wasting in at-risk populations such as those with alcohol use disorder.


PROX1. This manuscript reviews the molecular mechanism of development, integrity maintenance, and junction transformation in lacteal related to obesity.Lacteals are the central lymphatic vessels in the villi of the small intestine and perform nutrient absorption, especially dietary lipids, and the transportation of antigen and antigen-presenting cells. Remodeling, proliferation, and cell-cell junctions of lymphatic endothelial cells (LECs) in lacteals are the basis of the maintenance of lacteal integrity and dietary lipid absorption. Normal lipid absorption in the diet depends on sound lacteal development and proliferation, especially integrity maintenance, namely, maintaining the appropriate proportion of button-like and zipper-like junctions. Maintaining the integrity and transforming button-to-zipper junctions in lacteals are strongly connected with obesity, which could be regulated by intestinal flora and molecular signalings, such as vascular endothelial growth factor C-vascular endothelial growth receptor 3 (VEGFC-VEGFR3) signaling, Hippo signaling, Notch signaling, angiopoietin-TIE signaling, VEGF-A/VEGFR2 signaling, and  Of thes2c serotonin agonists, opioid receptor antagonist/dopamine, noradrenaline reuptake inhibitor, pancreatic lipase inhibitor, and incretin co-agonists , lymphatic vessel endothelial hyaluronic acid receptor 1 (LYVE1), C–C motif chemokine 21 (CCL21), neuropilin‐2 (NRP2), and vascular endothelial growth factor receptor 3 (VEGFR3) are the specific markers in lymphatic endothelial cells (LECs) . At the c period . Specifivia the receptor VEGFR-3  signaling molecules, which could be produced by small intestinal endothelial cells . VEGFs i VEGFR-3 . In the  VEGFR-3 , and VEG VEGFR-3 . Further VEGFR-3 , 24. A p VEGFR-3 . In the Prox1+ LECs  induced abnormal lymphatic vessel morphology resulting from defective spouting and maturation of lymphatic vessels , which decreased VEGFR3 expression, resulting in cell apoptosis and growth inhibition  and tight junction proteins  organize button and zipper junctions  to be transported into enterocytes . In the At present, button-like junctions of lacteals are recognized as the channel that allows the entry of chylomicron , 60. In CD36 knockout mice, revealing that CD36 might facilitate cholesterol uptake in the small intestine -related signaling: Calcrl, a receptor complex of adrenomedullin (AM), is highly expressed in LECs and is necessary for lacteal integrity and function . Dilatedlacteals . Furtheral cells .Gut microbiota: The gut microbiota is an essential factor that regulates lacteals’ development, maturation, and integrity. Delayed lacteal growth in the postnatal stage, lacteal regression in adulthood, and reduced button-like junctions among LECs had been found in germ-depleted mice. Meanwhile, the depletion of gut microbiota reduced button-like junctions and elevated zipper-like junctions in lacteals, resulting in blocked absorption of dietary fat . Thus, bProx1+ LECs numbers were visible in lacteals of germ-free (GF) mice  mice . Furtherntegrity . Myeloidntegrity . Conditintestine . TherefoVegfc gene using the Cre-Lox system  and neuropilin1 (NRP1) deletion increased zipper-like junctions and impeded dietary lipid uptake, resulting in resistance to HFD-induced obesity. The molecular mechanism was that the absence of NRP1 and FLT1 receptors bumped up VEGF-A bioactivity and signaling through VEGFR2, accelerating lacteal junction zippering and defective chylomicron uptake. High VEGF-A signaling transformed button-like junctions into zipper-like junctions, making lacteal junctions more compact concomitantly with reduced lipid absorption to prevent mice from HFD-induced obesity .In accordance with the above study, activation of VEGF-A/VEGFR2 signaling promotes the transformation of button-to-zipper junctions to reduce lipid uptake, which is resistant to obesity , 60, 67.DLL4 at the gene level induced continuous VE-cadherin junctions in LECs and significantly impaired lipid uptake and transportation in lacteal tissue  from defective lymphatic vessels  in clinical studies. A European cross-sectional study showed that single nucleotide polymorphisms (SNPs) in Prox1 were involved in genetic susceptibility to T2DM  have been advanced to human trials . RegulatIn the clinic, lacteal dysfunction results in defective nutrient absorption, such as protein-loss enteropathy-induced systemic lymphopenia, hypoalbuminemia, and hypogammaglobulinemia, because of serum protein loss in the gastrointestinal tract . In famiProx1 have been confirmed to be related to obesity. Enhancing the transformation of button-to-zipper junctions and attenuating VEGF-C levels have been demonstrated to resist obesity in mouse models. The small intestine is a large endocrine organ that synthesizes and secretes hormones, such as peptide YY, glucagon-like peptide 1 (GLP-1), cholecystokinin, somatostatin, gastric inhibitory peptide, serotonin, and ghrelin, which function to regulate metabolism, namely, insulin secretion, nutrient absorption and food intake (VEGF-C signaling, the transformation of zipper-to-button junctions, and d intake , 85. GutQX and YG, writing - original draft, writing – review, and editing. MH and KF, visualization. HD and LX, resources and investigation. JG and FL, supervision and conceptualization. All authors contributed to the article and approved the submitted version.This study was supported by the National Natural Science Foundation of China, NO. 81904158, NO.82004200, NO. 81904010, and the National Key R&D Program of China, NO. 2018YFC1704200.Most icons in the figures are supported by Biorender, and the article has got access to the publication and licensing rights of Biorender.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The article by Shannon and co-workers in this issue2 This architecture allows to broaden considerably the spectrum of biological responses to a single extracellular agonist, still relying on a limited toolkit  of receptor subtypes and downstream signals. This magic has two main ingredients: (1) the receptor, the downstream signals, and the target are in close proximity within the nanodomain,3 thus fostering mutual interactions; (2) the mutual interactions set up negative feed-back loops, which constrain signal duration and diffusion, or positive ones, which boost local signaling , as in hypertrophic remodeling, may result in loss of such specificity.8Mostly with the advent of dynamic FRET-based methodologies, the original concept of β-AR activating intracellular signals with diffuse intracellular targets has evolved to that of a system of confined receptor–signal complexes (nanodomains), each modulating functional elements according to their subcellular localization.ignaling .2,4–6 Pa2 The multiplicity of AKAP isoforms allows to target the associated signaling complex to a specific PKA target. AKAPs disruption results in loss of signal confinement, possibly contributing to diffusion of sarcolemmal cAMP to deep targets  upon internalization of sarcolemmal β-AR.2“Kinase Anchoring Proteins,” AKAPs in the case of PKA signaling, are pivotal in clustering the receptor and its signaling molecules within a nanodomain.While such a “local control” architecture provides clear advantages in terms of versatility and resolution, it detracts from signal “amplification”  and “pleiotropy” , among the fundamental properties of receptor signaling. These properties have experimental support and would be structure-saving, because they reduce the number of elements needed to modulate global cell function. How can they be reconciled with the steadily emerging evidence of local control?9 An intracellular location has been reported for multiple receptors previously considered “sarcolemmal”; for these receptors, intracellular diffusion of the agonist (possibly hindered by its enzymatic degradation) may account for modulation of deep targets, with AKAPs granting preservation of local control. However, unlike signal amplification by freely diffusible molecules, local control implies a stiff relationship between the signaling complex and its target. Therefore, a disproportion between abundance of PKA and of the target to be phosphorylated can be hardly accommodated. This is the case of PKA phosphorylation of phospholamban (PLN), the latter being about 1000-fold more abundant than PKA.10Local control and signaling pleiotropy, including deep targets, can be reconciled by the existence of complete intracellular receptor nanodomains activated by intracellular agonist, whose access to the cytosolic compartment is granted either by intrinsic liposolubility, or by specific sarcolemmal transporters (OCT for norepinephrine).1 address this issue by combining a localized perfusion strategy and FRET-based technology to β1-AR signaling in native rabbit ventricular myocytes. They show that the PLN-targeted intracellular β1AR-PKA-AKAP7γ complex they recently identified9 is endowed with considerable mobility, exceeding that of PLN itself. This implies that each activated PKA unit jumps from one PLN to the next, hence accomplishing signal amplification as required by the unbalanced stoichiometry. This process involves cyclic binding–unbinding of the signaling complex from PLN, made possible by the loss of PLN affinity, once phosphorylated, for AKAP7γ. Notably, the signaling complex may “surf” on the sarcoplasmic-reticulum (SR) membrane, possibly because AKAP7γ dimerization limits its 3D diffusion. In mobility (FRAP) experiments, AKAP7γ and PLN only were tagged; therefore, there was no direct evidence concerning the other components of the β-AR-PKA signaling cascade. Nonetheless AKAP7γ mobility was accompanied by diffusion, from the local agonist perfusion site, of the functional effects of phosphorylation. Persistence of norepinephrine (NE) effects in the presence of sotalol, a membrane impermeant β-AR antagonist, lends substantial support to the view that intracellular β-ARs contribute to PLN modulation. Interestingly, under sotalol, NE effect linearly increased throughout the 90 s recording; this was in sharp contrast with the rapidly saturating (within 50 s) response observed in the absence of sotalol.1 Thus, distinct signaling modalities, one recruiting sarcolemmal and intracellular β-ARs and the other based on intracellular ones only, modulate PLN with remarkably different kinetics. In terms of mechanism, short-range modulation of the β-AR signal within the AKAP nanodomain  and pentameric (noninhibitory) forms, the latter stabilized by PKA-mediated phosphorylation. By reducing availability of the monomeric form, pentamers phosphorylation contributes to SERCA stimulation by PKAThe unexpected information emerging from detailed analysis of nanodomain signaling with advanced optical methodologies contributes to explain puzzling aspects of cell biology and pharmacology, identifies potential therapeutic targets, and may help in devising disease-specific strategies in the use of receptor antagonists.


Chronic neck pain is a common musculoskeletal disorder caused by overuse of neck and upper back muscles or poor posture, and it is commonly combined with a limited range of motion in the neck and shoulders. Most cases will recover within a few days; however, the symptoms often recur easily. Fu's subcutaneous needling (FSN) is a new therapeutic approach used to treat patients with chronic neck pain. However, there is no solid evidence to support the effectiveness of FSN on chronic neck pain and disability. n = 60) with chronic neck pain for more than 2 months with pain intensity scored by visual analog scale (VAS) more than five were enrolled in this trial. Participants were equally randomized into the FSN or transcutaneous electrical nerve stimulation (TENS) group who received interventions once a day on day 1, day 2, and day 4. They were assessed by outcome measurements during pre- and post-treatment and followed up for 15 days.  Participants (P < 0.001). The immediate effects were also observed as the pressure pain threshold increased in the FSN group on day 2 (P=0.006) and day 4 (P=0.023) after treatment, and tissue hardness decreased by FSN on day 1 and day 2 after treatment (both P < 0.001). FSN and TENS treatment improved neck disability and mobility; moreover, FSN promoted participants to receive better sleep quality, as determined by PSQI assessment (P=0.030). TENS had no benefit on sleep quality.  The VAS was immediately reduced in the FSN and TENS groups and sustained for 15 days of follow-up (all NCT03605576, registered on July 30, 2018. FSN was able to relieve pain and relax muscle tightness. Notably, FSN significantly improved neck disability and mobility and enhanced sleep quality. These findings demonstrated that FSN could be an effective alternative treatment option for patients with chronic neck pain. Clinical Trial Registration: ClinicalTrials.gov Identifier:  The number of people with neck pain increases daily, and it has become a serious social problem in modern life. Acute neck pain is caused by overuse of the neck and upper back muscles or poor posture or injury, and it is usually combined with a limited range of motion . SymptomWaiguan) and LI 11 (Quchi) has been reported as an effective treatment to manage chronic neck pain caused by myofascial trigger points (MTrPs) on the upper trapezius muscle [Treatments for chronic neck pain aim to relieve pain and recover functional disability. In general, rest, good posture, and intake of non-steroidal anti-inflammatory drugs are good options for managing neck pain . Howevers muscle . Deep drs muscle . Fu's sus muscle . FSN is s muscle , 10. TheIn the present study, we evaluated the effectiveness of FSN on chronic neck pain by measuring visual analog scale (VAS), pressure pain threshold (PPT), tissue hardness (TH) meter, neck range of motion (NROM), neck disability index (NDI), and Pittsburgh sleep quality index (PSQI) as outcome measurements. Three treatment sessions were performed on day 1, day 2, and day 4, with assessments before each treatment session and immediately after treatment, as well as on day 8 and day 15 for follow-up.NCT03605576). All patients had completed their informed consent to participate in this study, and the research was conducted in accordance with the principles of the Declaration of Helsinki.Subjects who participated in this study were enrolled from the Departments of Physical Medicine and Rehabilitation and Acupuncture in the China Medical University Hospital according to an open-label, randomized controlled trial. This study was approved by the Institutional Review Board of the China Medical University Hospital (CMUH107-REC2-031) and registered with ClinicalTrials.gov (Identifier: The inclusion criteria were based on (1) adults older than 20 years old; (2) having chronic neck pain for more than 2 months, as defined by the International Association of the Study of Pain, updated in 2011 , and VASParticipants were randomly divided into FSN as the experimental group or TENS as the control group by a raffle system and allocated to the FSN or TENS group . A totalParticipants in the experimental group were treated by using a disposable Fu's subcutaneous needle  on the radial aspect of the forearm extensors muscle. The needle was inserted into the subcutaneous layer with the whole needle body by holding the inserting device . The insWaiguan) and LI 11 (Quchi), according to the guidance of WHO. The treatment parameters were set to pulse width of 200 μs, frequency of 200 Hz, and continuous wave for 20 min.Participants in the TENS group were treated with transcutaneous electrical nerve stimulator , with the electrodes attached to acupoints TE 5  as Fischer's methods to evaluate PPT , 16. FirSoft tissue stiffness was measured by using a tissue hardness meter  and applied in clinical studies recently –19. The Cervical Range of Motion (CROM) instrument  was used to assess NROM –22. A grNeck disability index (NDI) was modified from the Oswestry Low Back Pain Index , and it PSQI is the most effective tool to evaluate sleep quality in adults , 26. TheP < 0.05) among the results were calculated by using Statistical Package for Social Science (SPSS 18.0) for Windows. All data were expressed as mean ± standard deviation (SD). Baseline characteristics analysis of age, sex, VAS, PTT, TH, NROM, NDI, and PSQI was conducted via Student's t-test. For inferential statistics, the within-group analysis of all variables was conducted by paired sample t-test, whereas the between-group analysis of the variables was conducted by independent two-sample t-test.Statistically significant differences (P=0.870). The number of female participants was higher than the number of male participants in both groups . The affected side of the neck in the left and right sides were 17 and 13 participants for FSN and 14 and 16 participants for TENS, respectively. The VAS value was not significantly different in the FSN group compared with that in the TENS group . TH was similar in the two groups . No significant difference was observed in PPT  and NROM, including flexion, extension, left rotation, right rotation, left-side bending, and right-side bending in the two groups. Outcome assessments by questionnaire of NDI and PSQI showed no significant difference. No significant difference was found in all baseline values between the two groups. These results provide a well-randomized prospective study for further investigation.The baseline characteristics and outcome measurements of the two groups are shown in P < 0.001) and TH  significantly improved in the FSN group on day 1, except PPT . For the TENS group, VAS  and PTT  also significantly improved, except TH (pre-Tx: 56.80 ± 9.49 vs. post-Tx:54.06 ± 6.71). In the difference comparison (P < 0.001), not in PPT  and TH  compared with TENS. On day 2, VAS , PPT , and TH  significantly improved in the FSN group. However, TENS only significantly improved VAS , not PPT (P=0.738) and TH (P=0.596), on day 2. The comparison of differences revealed that FSN was more effective on VAS  and PPT  and TH  than TENS after the second course of treatment.To understand the immediate effect of FSN on chronic neck pain, we evaluated the VAS, PPT, and TH before and after treatment. The data are shown in mparison , FSN wasP < 0.001) and PPT  but not on TH (P=0.705). Only a significant effect on VAS  was observed in the TENS group. However, the difference between FSN and TENS in VAS, PPT, and TH was not significant.After an intermission of 1 day, the day 4 evaluation results showed that FSN still had a significant effect on VAS , whereas FSN had no significant effect on PTT. In addition, no significant decrease in TH was found in both groups on day 8 and day 15. Interestingly, TENS had a more significant decrease of PPT compared with FSN on day 8 and day 15 , and the effectiveness was sustained to day 15 . The effectiveness of TENS on NDI was from 10.16 ± 5.84 on day 1 pre-Tx to 8.33 ± 5.73 on day 8, P=0.036; and to 7.36 ± 5.70 on day 15, P=0.001. The two groups showed significant effects on short-term and long-term NDI assessment.Chronic neck pain commonly induces limitations of neck range of motion, followed by poor sleep quality. To estimate the effect of FSN on neck mobility, we analyzed the subjective questionnaire of NDI and objective NROM. The data in To understand further the effect of FSN on neck mobility, NROM was measured during pre- and post-treatment. Both FSN and TENS had benefits on neck motion upon treatment at days 1 and 2, except left rotation in the TENS group . After aP=0.504, and to 9.93 ± 2.74 on day 15, P=0.030; in the TENS group, from 10.80 ± 3.81 in pre-Tx day 1 to 10.26 ± 3.67 on day 8, P=0.290, and to 10.26 ± 3.18 on day 15, P=0.252; Improvement in sleep quality is an important outcome to assess the effectiveness of therapy. The results of the self-reported PSQI questionnaire indicated that FSN was beneficial for participants to achieve better sleep quality on day 15 follow-up compared with TENS  and TE 3 (Zhongzhu) is effective for acute neck pain caused by stiff neck or cervical spondylosis [Waiguan) and LI 11 (Quchi) are commonly used in acupuncture and TENS [Purpose-based acupoints are used for the treatment of various symptoms in ancient acupuncture theories. Needling on different acupoints produces distinct effects. For example, acupuncture at SI 3 , Supplementary Table 1 and Supplementary Table 2. Future research is required to establish a large population involving other institutes to amplify the statistical power and reach conclusive results.This study had some limitations. First, patients with chronic neck pain usually have an accompanying disability such as limited neck motion and poor sleep quality. The disorder is not restricted to elders only; up to 67% of the young population (aged 18–29 years) have had a 12-month prevalence of chronic neck pain . Young pThis study is the first to investigate FSN treatment of chronic neck pain with scientific evidence by using several objective evaluation tools in a clinical setting. FSN could not only relieve neck pain but also it improved the PPT and TH. Notably, FSN significantly improved neck disability and enhanced sleep quality.


An investigation of the ankle dynamics in a motor task may generate insights into the etiology of chronic ankle instability (CAI). This study presents a novel application of recurrence quantification analysis (RQA) to examine the ankle dynamics during walking. We hypothesized that CAI is associated with changes in the ankle dynamics as assessed by measures of determinism and laminarity using RQA.We recorded and analyzed the ankle position trajectories in the frontal and sagittal planes from 12 participants with CAI and 12 healthy controls during treadmill walking. We used time-delay embedding to reconstruct the position trajectories to a phase space that represents the states of the ankle dynamics. Based on the phase space trajectory, a recurrence plot was constructed and two RQA variables, the percent determinism (%DET) and the percent laminarity (%LAM), were derived from the recurrence plot to quantify the ankle dynamics.p < 0.05. effect size = 0.86). This indicated that the ankle dynamics in individuals with CAI is less likely to remain in the same state. No significant results were found in the %DET or in the sagittal plane.In the frontal plane, the %LAM in the CAI group was significantly lower than that in the control group (A lower frontal-plane %LAM may reflect more frequent switching between different patterns of neuromuscular control states due to the instabilities associated with CAI. With further study and development, %LAM may have the potential to become a useful biomarker for CAI. A lateral ankle sprain is one of the most prevalent orthopedic and sports injuries that can lead to long-term complications   and 12 gender- and age-matched (within 2 years of difference) healthy controls  were conveniently recruited from a university campus in 2016 and 2017. More females responded to our recruitment than males. All participants with CAI had an initial ankle sprain that occurred 1 year before enrollment, had multiple episodes of ankle giving way in the past 6 months before enrollment, and scored 24 or lower in the Cumberland Ankle Instability Tool (CAIT) . Participants' ankle position change over time was captured using a 3D motion capture system . The sampling frequency was set at 100 Hz. The marker placement and procedures for creating an ankle model were based on our previous study  or the matched leg  in the sagittal plane (dorsi- and plantarflexion) and frontal plane (eversion and inversion) over 25 consecutive strides using RQA. For CAI participants who had bilateral instability, the more severe side (based on the CAIT score) was selected for analysis. A stride was defined as the period between two consecutive initial contacts of the same foot. Initial contact was determined as the time when the marker on the calcaneus changed its moving direction from forward to backward . In this study, the distance threshold was set at 10% of the mean distance of all pairwise points  and the percent laminarity (%LAM), were derived from the RP to quantify the ankle dynamics. The formulas used to calculate these two variables were presented in Marwan et al. . Brieflyt-tests. Before carrying out the t-tests, its underlying assumptions of normality and homogeneity of variance were tested using the Shapiro–Wilk's test and Leven's test, respectively. We found all these assumptions were held. All statistical tests were performed using SPSS v25 . The alpha level was set at 0.05. In addition, Cohen's d was calculated to determine the effect size of the results, with d = 0.2 indicating a small effect, 0.5 a medium effect, and 0.8 a large effect  =p = 0.6, Cohen's d = −0.03, a small effect. The %LAM was slightly higher in the control group than that in the CAI group, but the difference did not reach a statistical significance, t(22) = −1.17, p = 0.26, Cohen's d = −0.4, a small effect.We tested the hypothesis that CAI is associated with changes in the ankle dynamics as assessed by measures of determinism (%DET) and laminarity (%LAM) using RQA. This hypothesis was partially supported by the results based on %LAM in the frontal plane. The lower %LAM observed in participants with CAI suggested that their ankle dynamics were less likely to stay in a specific state compared to their healthy counterparts. This more frequent switching between states could reflect compensations for instabilities associated with control at the ankle joint. However, such between-group difference in %LAM was not observed in the sagittal plane. Because ankle sprains or giving ways most often occur in the frontal plane, it is sensible to find deviation in ankle dynamics in the frontal but not the sagittal plane in individuals with CAI. It is important to note that the RQA results were computed from the phase space trajectory, and therefore cannot be mapped directly onto kinematic fluctuations in the original ankle inversion-eversion trajectory. No significant results were found in %DET in both frontal and sagittal planes. This variable indicates whether the ankle dynamics moves through the phase space in a regular, predictable manner. Thus, researchers suggested that this variable may reflect the predictability of a dynamical system  is episodes of the ankle giving way, which has been defined as “the regular occurrence of uncontrolled and unpredictable episodes of excessive inversion of the rear foot, which do not result in an acute lateral ankle sprain”  and laminarity (%LAM) using RQA. The %LAM was able to detect a change in ankle dynamics in individuals with CAI during walking. This result may reflect more frequent switching between different patterns of neuromuscular control states due to instabilities associated with CAI. With further study and development, this measure may have the potential to become a useful biomarker for CAI.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Subject Research Protection, Northeastern University. The patients/participants provided their written informed consent to participate in this study.S-CY conceived and designed research. EF collected data. SQ, CH, and C-AC contributed analytical tools. S-CY, EF, SQ, CH, and C-AC analyzed and interpreted data. S-CY drafted the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.