Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology最新文献

{"title":"The IP3-Dependent Signaling Pathway Is Involved in the Regulation of Atrophic Processes in Skeletal Muscles during Their Unloading","authors":"T. L. Nemirovskaya,&nbsp;K. A. Zaripova,&nbsp;S. P. Belova,&nbsp;K. A. Sharlo,&nbsp;D. A. Sidorenko","doi":"10.1134/S1990747825700473","DOIUrl":"10.1134/S1990747825700473","url":null,"abstract":"<p>The hypothesis of the relationship between changes in the calcium throughput of dihydropyridine receptors (DHPR) and the accumulation of ATP and phosphoinositide 3-kinases (PI3) in the muscle during its unloading was checked. We also checked the PI3K signaling pathway involvement in the atrophic process regulation in skeletal muscles during their unloading. To do this, we inhibited DHPR and PI3K with nifedipine and with LY294002. We found that both sarcolemma-related units are involved in the regulation of ATP content, calcium signaling markers, and IP3 levels in the unloaded <i>m. soleus</i>. DHPR inhibition prevents ATP, Ca²⁺, and IP3 accumulation in soleus under unloading. PI3K inhibition reduces the degree of atrophy, ATP and IP3 content in muscle, and regulates the Ca-dependent signaling markers under tree day soleus unloading.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"481 - 486"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510366","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Changes of the Structure and Permeability of Lipid Membranes Caused by Nanoparticles and Pulsed Electromagnetic Effects","authors":"Yu. V. Gulyaev,&nbsp;V. A. Cherepenin,&nbsp;I. V. Taranov,&nbsp;V. A. Vdovin,&nbsp;A. A. Yaroslavov,&nbsp;K. V. Potapenkov,&nbsp;G. B. Khomutov","doi":"10.1134/S1990747825700400","DOIUrl":"10.1134/S1990747825700400","url":null,"abstract":"&lt;p&gt;Studies of structure and permeability changes of biological and biomimetic lipid membranes caused by natural and artificial factors are currently an important field of fundamental science and applied research. A prominent interdisciplinary area of such research with high potential of biomedical applications is the development of new effective methods of drug therapy aimed at solving the problem of controlled addressed delivery of drugs and genes directly to target areas and cells of the body (spatial-temporal coupling and control of drug delivery). Our work is devoted to the development of effective and safe biocompatible means and methods of encapsulation, targeted delivery and controlled release of drugs in aqueous media including living systems. For capsulation of drug compounds in colloid carriers we use originally fabricated nanostructured biomimetic lipid membrane vesicles (nanocomposite liposomes) whose membranes are functionalized with magnetite and gold nanoparticles. To solve the problem of safe controlled release of a capsulated drug in the aqueous media we develop an approach based on the use of strong ultrashort electrical pulses with duration of less than 10 ns, providing the non-thermal effect of selective controlled electroporation of nanocomposite lipid membranes containing conductive nanoparticles polarized in applied external electric field. A theoretical model of non-thermal interaction of nanostructured liposomal capsules with ultrashort external electrical pulses has been developed, within the framework of which an expression has been obtained for the critical value of the applied electric field strength, which determines the threshold for the occurrence of the electroporation and decapsulation effect in a conducting aqueous medium. The key role of electrically conductive nanoparticles in increasing the sensitivity of the structure and conductivity of nanocomposite liposomes to external ultrashort electrical action is shown. The theoretically described mechanism of change in the structure and conductivity of lipid membranes containing electrically conductive nanoparticles explains the selective controlled nature of ultrashort pulse action on nanocomposite liposomal containers. Experimentally, the effect of controlled selective changes of permeability and decapsulation of nanocomposite liposomes in our work was recorded by conductometry methods in experiments with liposomes containing a concentrated NaCl solution inside, as well as by fluorimetry methods in experiments with the anticancer antibiotic doxorubicin and fluorescent dye carboxyfluorescein, which were loaded into liposomal carriers as model molecular compounds. Encapsulated payload was released from nanocomposite liposomes after the influence of short strong electric pulses with efficiency up to 98%. The data on membrane permeability changes correlated well with results on structural changes of nanocomposite liposomes registered by transmission electron microscop","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"423 - 432"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510445","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Receptor Histidine Kinases in Green Algae: Domain Analysis","authors":"E. V. Laptenkova,&nbsp;E. V. Zinovev,&nbsp;V. I. Gordeliy","doi":"10.1134/S1990747825700424","DOIUrl":"10.1134/S1990747825700424","url":null,"abstract":"<p>In bacteria and some eukaryotes, an important tool to respond to both external and internal signals is a two-component system, which is typically composed of a transmembrane receptor histidine kinase (RHK) and its cognate response regulator. While architecture and distribution of RHKs in prokaryotes are well-documented, their roles in eukaryotes, particularly in the green algae, remain less understood. Here, we analyzed the sequences and domain architectures of RHKs within the green algae clade <i>Chlorophyta</i>, in the class <i>Chloropicophycea</i>e, better characterized compared to other classes. Our study aims to deepen understanding and infer the biological roles of these proteins. Based on InterProScan results, we showed that rhodopsin-histidine kinases play a global role in the biology of the microalgae: they dominate among all RHKs, representing 57% of them. The length variation of RHKs does not have a discrete structure. Furthermore, we identified the most frequent domains and domain architectures in the proteins, with a notable discovery of a significant number of hybrid histidine kinases, where the sensory domain is a microbial rhodopsin protein. We demonstrate also that one-half of the identified rhodopsin-histidine kinases possess a catalytic domain of either adenylyl or guanylyl cyclase, making them potentially useful as optogenetic tools. In addition, it is shown that a significant part of RHKs in green algae contain not one but two REC-domains. These findings, along with previous reports, suggest that multiple REC-domains may support complex regulatory functions, possibly allowing condition-dependent responses. Our work may also help to understand better RHKs potential in optogenetics.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"440 - 448"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tardigrade Dsup Protein Protects Human Cells during Proton Radiotherapy","authors":"A. V. Rzyanina,&nbsp;E. V. Kravchenko,&nbsp;M. P. Zarubin,&nbsp;G. V. Mytsin","doi":"10.1134/S1990747825700485","DOIUrl":"10.1134/S1990747825700485","url":null,"abstract":"<p>Principles of resistance to physicochemical stresses in extremophile organisms are unique for the development of new protective technologies. In this work, Dsup (Damage suppressor) protein, which belongs to one of the most radiation-resistant animals—tardigrade <i>Ramazzottius varieornatus</i>, was studied as a perspective radioprotector for proton therapy. Dsup, known for its effective DNA-protective properties, was produced in human cells HEK293 expressing <i>Dsup</i> gene. To study radio resistance, human cells were irradiated by high-energy protons (150 MeV) at Medical-Technical Complex (MTC) of Phasatron (JINR). Within the actual medical protocol of proton radiation therapy, Dsup was confirmed to be the potential protector for healthy cells and tissues.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"487 - 490"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510360","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling the Distribution Kinetics of Charged Drugs in Tumor Cells under Variable Electric Fields Gradients on Mitochondrial and Nuclear Membranes","authors":"K. Z. Askarova-Kaspirovich,&nbsp;G. I. Morozova","doi":"10.1134/S1990747825700357","DOIUrl":"10.1134/S1990747825700357","url":null,"abstract":"<p>The distribution of charged cytostatics or photosensitizers (PS) in tumor cells (TC) is an important factor in anticancer therapy effectiveness. The aim of our study was to build the mathematical model of the distribution kinetics of charged PSs, namely, chlorin e6 (E6^‒) and its derivative dimethyl ether (DME⁺) in TC with variable electric transmembrane potentials (TMPs) on mitochondrial and nuclear membranes. A kinetic model is presented that includes a system of four nonlinear differential equations describing the accumulation of PS in a certain model system based on the Nernst theory. This system consists of compartments separated by membranes with different TMPs. The sum of these potentials includes negative plasma and mitochondrial TMPs, as well as an energy-dependent positive TMP on the inner nuclear membrane. Numerical solutions of the nonlinear system of equations with given modulation parameters of TMPs were obtained using a special computer program. The initial rates of PSs transfer and membrane permeability were determined using the curves of fluorescence intensity changes of E6^‒ and DME⁺ in lymphoblast cells. Comparative analysis of the data showed that the effective accumulation of the charged drugs depended on the optimal TMPs ratio in tumor cells. These TMPs can be determined using voltage sensitive fluorescent cationic probe 4-(<i>p</i>-di-methylaminostyryl)-1-methylpyridinium (DSM⁺).</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"392 - 397"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PCMM Nanomaterials as Multifunctional Bioprobes Studied in Animal Model of C. elegans","authors":"E. Marusich,&nbsp;M. Nikiforova,&nbsp;E. Stepanidenko,&nbsp;I. Arefina,&nbsp;A. Yashenok","doi":"10.1134/S199074782570045X","DOIUrl":"10.1134/S199074782570045X","url":null,"abstract":"<p>Multifunctional nanomaterials which are active in the near infrared (NIR) region and can be physically guided (laser light, magnetic field, ultrasound, etc.) hold a great promise in several biomedical applications, such as drug delivery, cell biology, biosensing, and bioimaging. In this study, we have developed multifunctional photoluminescence coding magnetic microspheres (PCMMs), studied their physical and chemical properties, and explored the possibility of using PCMMs in living biological organisms. To reach this goal, we investigated the possibility of PCMM imaging in the <i>C. elegance</i> animal model. We performed extensive toxicity screening of 10 types of luminescent magnetic microspheres (LMMs) and 5 types of carbon dots (CDs), which were embedded into LMM. We also explored the delivery and localization of tested nanoparticles inside the nematode body. It was found that the particles we studied are not toxic to living <i>C. elegance</i> tissue in the optimized concentration range and tend to extend the lifespan of nematodes. Fluorescent microscopy studies revealed the localization of CDs and LMMs in the intestinal part of the nematode body. Our results demonstrated the possibility of detecting photoluminescent PCMM magnetic microspheres in living organisms, implying the potential of PCMM for the development of this nanoscale drug delivery system for future human studies.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"465 - 473"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Study of Complexation of AED Peptide and Lysine Based Dendrimer with HisArg Spacers","authors":"S. E. Mikhtaniuk,&nbsp;V. V. Bezrodnyi,&nbsp;A. Y. Vakulyuk,&nbsp;I. M. Neelov,&nbsp;O. V. Shavykin","doi":"10.1134/S1990747825700461","DOIUrl":"10.1134/S1990747825700461","url":null,"abstract":"<p>This study explores the interaction of bioactive AED tripeptide molecules with new lysine-based dendrimer KHR containing spacers consisting of two different amino acid residues (histidine-arginine, HR). Previous research has examined second-generation lysine dendrimers containing double charged lysine-lysine and arginine-arginine spacers as well as double hydrophobic alanine-alanine and leucine-leucine, and double pH-sensitive histidine-histidine spacers. Interactions of some of these dendrimers with several bioactive peptide, for example, AEDG were studied also. In this work, similar molecular dynamics simulations were conducted to study the complexation of 16 AED molecules with KHR dendrimer at two pH values: (a) pH &gt; 7 with uncharged histidines and (b) pH &lt; 5 with fully protonated histidines. Results indicate that dendrimer at both pH make complex with AED but dendrimer at smaller pH can accommodate more AED molecules.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"474 - 480"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145511051","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biofilms of MDR K. pneumoniae (hvKp) Strains Can be Destroyed by Free Fatty Acids Interactions with Bacterial Cell Wall Membranes","authors":"E. I. Marusich,&nbsp;H. Mohamed","doi":"10.1134/S1990747825700448","DOIUrl":"10.1134/S1990747825700448","url":null,"abstract":"<p>Hypervirulent mucoviscus <i>Klebsiella pneumoniae</i> (hvKp) is rapidly emerging as opportunistic pathogens that have a global impact leading to a significant increase in mortality rates among clinical patients. Anti-virulence strategies that target bacterial adhesion and biofilm formation, proposed as alternatives to antibiotic treatments for reducing the rapid emergence of bacterial resistance. The main objective of this study was to examine the efficacy of fatty acid-enriched extract (AWME3) derived from the fat of <i>Hermetia illucens</i> fly larvae to combat biofilms formed by multidrug-resistant (MDR) and highly virulent hvKp pathogens. To reach these goals we used crystal violet (CV) and ethidium bromide (EtBr) assays on the mature biofilms of <i>K. pneumoniae</i> KPi1627, KPM9 and ATCC BAA-2473 strains. We found that exposure of hvKp strains to AWME3 at a concentration of 500 µg/mL (corresponding to 2× minimum inhibitory concentration, MIC) significantly affects the membrane permeability (<i>p</i> &lt; 0.0001), causing serious disturbances and changes in the composition of membrane phospholipids, as confirmed by the detected increase in CV and EtBr uptake by 70 and 80%, respectively, compared to the control group. We used scanning electron microscopy (SEM) as a direct microscopic method to collect evidence of bactericidal action of AWME3 extract and the destruction of hvKp biofilms. In conclusion, our study demonstrates the exceptional capability of natural AWME3 extract, enriched with a unique combination of fatty acids, to effectively eliminate biofilms formed by highly drug-resistant and highly virulent pathogens <i>K. pneumoniae</i> (hvKp). Our results highlight the potential to control and minimize the rapid emergence of bacterial resistance by treating biofilm-associated infections caused by hvK pathogens with AWME3.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"456 - 464"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510365","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Kinetic Characteristics of Erythrocyte Membrane Acetylcholinesterase in Ground Squirrels During Hibernation and Arousal","authors":"A. M. Dzhafarova,&nbsp;E. V. Vlasova,&nbsp;N. K. Klichkhanov","doi":"10.1134/S1990747825700382","DOIUrl":"10.1134/S1990747825700382","url":null,"abstract":"<p>Hibernation of small mammals is unique in that it consists of bouts—alternating periods of deep hibernation and inter-bout arousals, during which the animals warm up and many body parameters return to the euthermic level. Significant fluctuations in body temperature during the transition of an animal from a torpid state to a euthermic one can affect membrane-bound enzymes. In this work, the activity and kinetic parameters of acetylcholinesterase (AChE) in erythrocyte membranes of small ground squirrels (<i>Spermophilus pygmaeus</i> Pall.) during deep torpor and in the dynamics of exit from it was studied. The activity of AChE in the membranes of ground squirrel erythrocytes was determined by Ellman’s method. The kinetic characteristics of AChE (maximum velocity (<i>V</i>_m), Michaelis constant (<i>K</i>_m) and substrate inhibition constant (<i>K</i>_i)) were found by the least squares method in accordance with the Haldane model. The study showed that in the torpid state, the activity and <i>V</i>_m of AChE do not change significantly. At the same time, <i>K</i>_m decreases and <i>K</i>_i increases, which contributes to a significant change in the character of the concentration dependence of AChE and a decrease in the degree of substrate inhibition. During arousal of ground squirrels from hibernation at Tb 25°C, <i>V</i>_m increases significantly, which sharply increases the efficiency of AChE catalysis, despite the fact that the <i>K</i>_m value also increases. After the final warming of the ground squirrels from hibernation (Tb 37°C), the values of <i>V</i>_m and <i>K</i>_m of AChE and, accordingly, the efficiency of catalysis do not normalize but remain at the level achieved at Tb 25°C. In this case, <i>K</i>_i values decrease relative to hibernation, which increases the degree of substrate inhibition and narrows the range of effective acetylcholine concentrations. The detected sharp changes in the activity of AChE of ground squirrel erythrocytes AChE during awakening can play an important role in the rapid decrease in the level of circulating acetylcholine and the improvement of peripheral microcirculation.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"410 - 415"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510362","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Changes in Electrical Properties of Bilayer Lipid Membranes Induced by Colloidal Magnetite (Fe3O4) Nanoparticles in a Static Inhomogeneous Magnetic Field","authors":"E. D. Borisova,&nbsp;A. A. Anosov,&nbsp;A. A. Bolshakova,&nbsp;E. Yu. Smirnova,&nbsp;I. V. Taranov,&nbsp;G. B. Khomutov,&nbsp;I. V. Grigorian,&nbsp;V. V. Spiridonov,&nbsp;V. A. Cherepenin","doi":"10.1134/S1990747825700370","DOIUrl":"10.1134/S1990747825700370","url":null,"abstract":"<p>The changes of electrical parameters of azolectin membranes in a static inhomogeneous magnetic field at the one-sided addition of positively charged quasi-spherical superparamagnetic magnetite nanoparticles (MNPs) with a diameter of about 4 nm were studied. The magnet was located at different distances from the membrane, and nanoparticles were attracted to the membrane surface with different strengths by the magnetic field. After the addition of MNPs, a decrease in capacitance, polarization, and further depolarization of the membranes was observed. Zero current appeared on the bilayer lipid membranes (BLMs). There was no unambiguous dependence of the zero current on the membrane depolarization and there were no dependences of the zero current on the strength of the magnetic field and on the concentration of added MNPs. The membrane conductivity increased on average with an increase in the external magnetic field and depended on the ionic strength. Magnetic field caused a rapid accumulation of MNPs on the surface of the BLMs. As a result, the membrane was polarized. Depolarization of the membranes occurred when MNPs passed through the BLMs. The increase in conductivity reflected the processes of lipid bilayer disordering.</p>","PeriodicalId":484,"journal":{"name":"Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology","volume":"19 :","pages":"404 - 409"},"PeriodicalIF":1.4,"publicationDate":"2025-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145510364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}