Pflugers Archiv : European journal of physiology最新文献

{"title":"Establishment of the deuterium oxide dilution method as a new possibility for determining the transendothelial water permeability.","authors":"Hannes Müller, Janina Hahn, Angelina Gierke, Robert Stark, Cornelia Brunner, Thomas K Hoffmann, Jens Greve, Oliver Wittekindt, Robin Lochbaum","doi":"10.1007/s00424-024-02934-z","DOIUrl":"10.1007/s00424-024-02934-z","url":null,"abstract":"<p><p>Increase in transendothelial water permeability is an essential etiological factor in a variety of diseases like edema and shock. Despite the high clinical relevance, there has been no precise method to detect transendothelial water flow until now. The deuterium oxide (D₂O) dilution method, already established for measuring transepithelial water transport, was used to precisely determine the transendothelial water permeability. It detected appropriate transendothelial water flow induced by different hydrostatic forces. This was shown in four different endothelial cell types. The general experimental setup was verified by gravimetry and absorbance spectroscopy. Determination of transendothelial electrical resistance (TEER) and immunocytochemical staining for proteins of the cell-cell contacts were performed to ensure that no damage to the endothelium occurred because of the measurements. Furthermore, endothelial barrier function was modulated. Measurement of transendothelial water flux was verified by measuring the TEER, the apparent permeability coefficient and the electrical capacity. The barrier-promoting substances cyclic adenosine monophosphate and iloprost reduced TEER and electrical capacity and increased permeability. This was accompanied by a reduced transendothelial water flux. In contrast, the barrier-damaging substances thrombin, histamine and bradykinin reduced TEER and electrical capacity, but increased permeability. Here, an increased water flow was shown. This newly established in vitro method for direct measurement of transendothelial water permeability was verified as a highly precise technique in various assays. The use of patient-specific endothelial cells enables individualized precision medicine in the context of basic edema research, for example regarding the development of barrier-protective pharmaceuticals.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139723/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140028709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Oscillatory work and the step that generates force in single myofibrils from rabbit psoas.","authors":"Masataka Kawai, Bogdan Iorga","doi":"10.1007/s00424-024-02935-y","DOIUrl":"10.1007/s00424-024-02935-y","url":null,"abstract":"<p><p>The elementary molecular step that generates force by cross-bridges (CBs) in active muscles has been under intense investigation in the field of muscle biophysics. It is known that an increase in the phosphate (P_i) concentration diminishes isometric force in active fibers, indicating a tight coupling between the force generation step and the P_i release step. The question asked here is whether the force generation occurs before P_i release or after release. We investigated the effect of P_i on oscillatory work production in single myofibrils and found that P_i-attached state(s) to CBs is essential for its production. Oscillatory work is the mechanism that allows an insect to fly by beating its wings, and it also has been observed in skeletal and cardiac muscle fibers, implying that it is an essential feature of all striated muscle types. With our studies, oscillatory work disappears in the absence of P_i in experiments using myofibrils. This suggests that force is generated during a transition between steps of oscillatory work production, and that the states involved in force production must have P_i attached. With sinusoidal analysis, we obtained the kinetic constants around the P_i release steps, established a CB scheme, and evaluated force generated (and supported) by each CB state. Our results demonstrate that force is generated before P_i is released, and the same force is maintained after P_i is released. Stretch activation and/or delayed tension can also be explained with this CB scheme and forms the basis of force generation and oscillatory work production.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140336500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The extracellular matrix protein fibronectin promotes metanephric kidney development.","authors":"Kathrin Skoczynski, Andre Kraus, Christoph Daniel, Maike Büttner-Herold, Kerstin Amann, Mario Schiffer, Kristina Hermann, Leonie Herrnberger-Eimer, Ernst R Tamm, Bjoern Buchholz","doi":"10.1007/s00424-024-02954-9","DOIUrl":"10.1007/s00424-024-02954-9","url":null,"abstract":"<p><p>Complex interactions of the branching ureteric bud (UB) and surrounding mesenchymal cells during metanephric kidney development determine the final number of nephrons. Impaired nephron endowment predisposes to arterial hypertension and chronic kidney disease. In the kidney, extracellular matrix (ECM) proteins are usually regarded as acellular scaffolds or as the common histological end-point of chronic kidney diseases. Since only little is known about their physiological role in kidney development, we aimed for analyzing the expression and role of fibronectin. In mouse, fibronectin was expressed during all stages of kidney development with significant changes over time. At embryonic day (E) 12.5 and E13.5, fibronectin lined the UB epithelium, which became less pronounced at E16.5 and then switched to a glomerular expression in the postnatal and adult kidneys. Similar results were obtained in human kidneys. Deletion of fibronectin at E13.5 in cultured metanephric mouse kidneys resulted in reduced kidney sizes and impaired glomerulogenesis following reduced cell proliferation and branching of the UB epithelium. Fibronectin colocalized with alpha 8 integrin and fibronectin loss caused a reduction in alpha 8 integrin expression, release of glial-derived neurotrophic factor and expression of Wnt11, both of which are promoters of UB branching. In conclusion, the ECM protein fibronectin acts as a regulator of kidney development and is a determinant of the final nephron number.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139724/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140336501","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The pain target Na_V1.7 is expressed late during human iPS cell differentiation into sensory neurons as determined in high-resolution imaging.","authors":"Yi Liu, Rachna Balaji, Marcelo A Szymanski de Toledo, Sabrina Ernst, Petra Hautvast, Aylin B Kesdoğan, Jannis Körner, Martin Zenke, Anika Neureiter, Angelika Lampert","doi":"10.1007/s00424-024-02945-w","DOIUrl":"10.1007/s00424-024-02945-w","url":null,"abstract":"<p><p>Human-induced pluripotent stem cells (iPS cells) are efficiently differentiated into sensory neurons. These cells express the voltage-gated sodium channel Na_V1.7, which is a validated pain target. Na_V1.7 deficiency leads to pain insensitivity, whereas Na_V1.7 gain-of-function mutants are associated with chronic pain. During differentiation, the sensory neurons start spontaneous action potential firing around day 22, with increasing firing rate until day 40. Here, we used CRISPR/Cas9 genome editing to generate a HA-tag Na_V1.7 to follow its expression during differentiation. We used two protocols to generate sensory neurons: the classical small molecule approach and a directed differentiation methodology and assessed surface Na_V1.7 expression by Airyscan high-resolution microscopy. Our results show that maturation of at least 49 days is necessary to observe robust Na_V1.7 surface expression in both protocols. Electric activity of the sensory neurons precedes Na_V1.7 surface expression. A clinically effective Na_V1.7 blocker is still missing, and we expect this iPS cell model system to be useful for drug discovery and disease modeling.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139713/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140306431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Locally applied heat stress during exercise training may promote adaptations to mitochondrial enzyme activities in skeletal muscle.","authors":"Ed Maunder, Andrew King, Jeffrey A Rothschild, Matthew J Brick, Warren B Leigh, Christopher P Hedges, Troy L Merry, Andrew E Kilding","doi":"10.1007/s00424-024-02939-8","DOIUrl":"10.1007/s00424-024-02939-8","url":null,"abstract":"<p><p>There is some evidence for temperature-dependent stimulation of mitochondrial biogenesis; however, the role of elevated muscle temperature during exercise in mitochondrial adaptation to training has not been studied in humans in vivo. The purpose of this study was to determine the role of elevating muscle temperature during exercise in temperate conditions through the application of mild, local heat stress on mitochondrial adaptations to endurance training. Eight endurance-trained males undertook 3 weeks of supervised cycling training, during which mild (~ 40 °C) heat stress was applied locally to the upper-leg musculature of one leg during all training sessions (HEAT), with the contralateral leg serving as the non-heated, exercising control (CON). Vastus lateralis microbiopsies were obtained from both legs before and after the training period. Training-induced increases in complex I (fold-change, 1.24 ± 0.33 vs. 1.01 ± 0.49, P = 0.029) and II (fold-change, 1.24 ± 0.33 vs. 1.01 ± 0.49, P = 0.029) activities were significantly larger in HEAT than CON. No significant effects of training, or interactions between local heat stress application and training, were observed for complex I-V or HSP70 protein expressions. Our data provides partial evidence to support the hypothesis that elevating local muscle temperature during exercise augments training-induced adaptations to mitochondrial enzyme activity.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139708/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140040070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Acute sodium bicarbonate administration improves ventilatory efficiency in experimental respiratory acidosis: clinical implications.","authors":"Horacio J Adrogué, Nicolaos E Madias","doi":"10.1007/s00424-024-02949-6","DOIUrl":"10.1007/s00424-024-02949-6","url":null,"abstract":"<p><p>Administering sodium bicarbonate (NaHCO₃) to patients with respiratory acidosis breathing spontaneously is contraindicated because it increases carbon dioxide load and depresses pulmonary ventilation. Nonetheless, several studies have reported salutary effects of NaHCO₃ in patients with respiratory acidosis but the underlying mechanism remains uncertain. Considering that such reports have been ignored, we examined the ventilatory response of unanesthetized dogs with respiratory acidosis to hypertonic NaHCO₃ infusion (1 N, 5 mmol/kg) and compared it with that of animals with normal acid-base status or one of the remaining acid-base disorders. Ventilatory response to NaHCO₃ infusion was evaluated by examining the ensuing change in PaCO₂ and the linear regression of the PaCO₂ vs. pH relationship. Strikingly, PaCO₂ failed to increase and the ΔPaCO₂ vs. ΔpH slope was negative in respiratory acidosis, whereas PaCO₂ increased consistently and the ΔPaCO₂ vs. ΔpH slope was positive in the remaining study groups. These results cannot be explained by differences in buffering-induced decomposition of infused bicarbonate or baseline levels of blood pH, PaCO₂, and pulmonary ventilation. We propose that NaHCO₃ infusion improved the ventilatory efficiency of animals with respiratory acidosis, i.e., it decreased their ratio of total pulmonary ventilation to carbon dioxide excretion (V_E/V_CO2). Such exclusive effect of NaHCO₃ infusion in animals with respiratory acidosis might emanate from baseline increased V_D/V_T (dead space/tidal volume) caused by bronchoconstriction and likely reduced pulmonary blood flow, defects that are reversed by alkali infusion. Our observations might explain the beneficial effects of NaHCO₃ reported in patients with acute respiratory acidosis.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140294186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Electronic cigarettes and cardiovascular disease: epidemiological and biological links.","authors":"Huiqi Zong, Zhekai Hu, Weina Li, Mina Wang, Qi Zhou, Xiang Li, Hongxu Liu","doi":"10.1007/s00424-024-02925-0","DOIUrl":"10.1007/s00424-024-02925-0","url":null,"abstract":"<p><p>Electronic cigarettes (e-cigarettes), as alternative nicotine delivery methods, has rapidly increased among youth and adults in recent years. However, cardiovascular safety is an important consideration regarding e-cigarettes usage. e-cigarette emissions, including nicotine, propylene glycol, flavorings, nitrosamine, and metals, might have adverse effects on cardiovascular health. A large body of epidemiological evidence has indicated that e-cigarettes are considered an independent risk factor for increased rates of cardiovascular disease occurrence and death. The incidence and mortality of various types of cardiovascular disease, such as cardiac arrhythmia, hypertension, acute coronary syndromes, and heart failure, have a modest growth in vapers (users of e-cigarettes). Although the underlying biological mechanisms have not been fully understood, studies have validated that oxidative stress, inflammation, endothelial dysfunction, atherosclerosis, hemodynamic effects, and platelet function play important roles in which e-cigarettes work in the human body. This minireview consolidates and discusses the epidemiological and biological links between e-cigarettes and various types of cardiovascular disease.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11139732/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139906312","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Heat production during exercise in pregnancy: discerning the contribution of total body weight.","authors":"Nicholas O'Rourke, Sheila Dervis, Danilo F da Silva, Carla Geurts, François Haman, Kristi Bree Adamo","doi":"10.1007/s00424-024-02929-w","DOIUrl":"10.1007/s00424-024-02929-w","url":null,"abstract":"<p><p>Studies have reported enhanced thermoregulatory function as pregnancy progresses; however, it is unclear if differences in thermoregulation are attributed to weight gain or other physiological changes. This study aimed to determine if total body weight will influence thermoregulation (heat production (H_prod)), heart rate, and perceptual measurements in response to weight-bearing exercise during early to late pregnancy. A cross-sectional design of healthy pregnant women at different pregnancy time points (early, T1; middle, T2; late, T3) performed a 7-stage weight-bearing incremental exercise protocol. Measurements of H_prod, HR, and RPE were examined. Two experimental groups were studied: (1) weight matched and (2) non-weight matched, in T1, T2, and T3. During exercise, equivalent H_prod at T1 (326 ± 88 kJ), T2 (330 ± 43 kJ), and T3 (352 ± 52 kJ) (p = 0.504); HR (p = 0.830); and RPE (p = 0.195) were observed in the WM group at each time point. In the NWM group, H_prod (from stages 1-6 of the exercise) increased across pregnancy time points, T1 (291 ± 76 kJ) to T2 (347 ± 41 kJ) and T3 (385 ± 47 kJ) (p < 0.001). HR increased from T1 to T3 in the warm-up to stage 6 (p = 0.009). RPE did not change as pregnancy time point progressed (p = 0.309). Total body weight, irrespective of pregnancy time point, modulates H_prod and HR during exercise. Therefore, accounting for total body weight is crucial when comparing thermoregulatory function during exercise across pregnancy.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140022323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.","authors":"Wataru Kudo, Hikaru Hashitani","doi":"10.1007/s00424-024-02931-2","DOIUrl":"10.1007/s00424-024-02931-2","url":null,"abstract":"<p><p>Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca²⁺-activated K⁺ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca²⁺ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca²⁺ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca²⁺ spikes during each burst of spontaneous Ca²⁺ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca²⁺ release which prematurely terminates Ca²⁺ transient bursts resulting in the attenuation of SPCs.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139990866","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biophysical mechanisms of myocardium sodium channelopathies.","authors":"Anastasia K Zaytseva, Olga E Kulichik, Anna A Kostareva, Boris S Zhorov","doi":"10.1007/s00424-024-02930-3","DOIUrl":"10.1007/s00424-024-02930-3","url":null,"abstract":"<p><p>Genetic variants of gene SCN5A encoding the alpha-subunit of cardiac voltage-gated sodium channel Na_v1.5 are associated with various diseases, including long QT syndrome (LQT3), Brugada syndrome (BrS1), and progressive cardiac conduction disease (PCCD). In the last decades, the great progress in understanding molecular and biophysical mechanisms of these diseases has been achieved. The LQT3 syndrome is associated with gain-of-function of sodium channels Na_v1.5 due to impaired inactivation, enhanced activation, accelerated recovery from inactivation or the late current appearance. In contrast, BrS1 and PCCD are associated with the Na_v1.5 loss-of-function, which in electrophysiological experiments can be manifested as reduced current density, enhanced fast or slow inactivation, impaired activation, or decelerated recovery from inactivation. Genetic variants associated with congenital arrhythmias can also disturb interactions of the Na_v1.5 channel with different proteins or drugs and cause unexpected reactions to drug administration. Furthermore, mutations can affect post-translational modifications of the channels and their sensitivity to pH and temperature. Here we briefly review the current knowledge on biophysical mechanisms of LQT3, BrS1 and PCCD. We focus on limitations of studies that use heterologous expression systems and induced pluripotent stem cells (iPSC) derived cardiac myocytes and summarize our understanding of genotype-phenotype relations of SCN5A mutations.</p>","PeriodicalId":19954,"journal":{"name":"Pflugers Archiv : European journal of physiology","volume":null,"pages":null},"PeriodicalIF":4.5,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139997120","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}