Biomedical Microdevices最新文献

{"title":"Fast Sub-Hz potentiostatic/galvanostatic bio-impedance measurements using chaotic oscillators","authors":"Abdulwadood Al-Ali,&nbsp;Ahmed Elwakil,&nbsp;Brent Maundy,&nbsp;Sohaib Majzoub","doi":"10.1007/s10544-022-00641-z","DOIUrl":"10.1007/s10544-022-00641-z","url":null,"abstract":"<div><p>The measurement of bio-impedance spectra at ultra low frequencies (sub-Hz) is known to require a considerably long time with the classical frequency-sweep method or other narrow-band periodic excitation signals. In this work, an impedance measurement technique based on using wide-band chaotic signals is proposed and experimentally validated over the frequency range <span>(10,mHz-1,Hz)</span>. The technique was tested in both potentiostatic and galvanostatic modes, first using commercial components and then using an enhanced Howland current pump designed and fabricated in a 65nm CMOS technology. The accuracy of the proposed technique was assessed on fruit samples compared to measurements conducted using a research-grade Biologic VSP-300 electro-chemical station.</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4402986","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":"An automated microfluidic system with one-dimensional beads array for multiplexed torch detection at point-of-care testing","authors":"Hao Li,&nbsp;Shengda Yu,&nbsp;Dong Wang,&nbsp;Xinying Huang,&nbsp;Qiang Fu,&nbsp;Donglin Xu,&nbsp;Lulu Zhang,&nbsp;Shizhi Qian,&nbsp;Xianbo Qiu","doi":"10.1007/s10544-022-00629-9","DOIUrl":"10.1007/s10544-022-00629-9","url":null,"abstract":"<div><p>An automated microfluidic system with functionalized beads has been developed for multiplexed TORCH detection at point-of-care testing. A concise microfluidic chip consisting of a one-dimensional beads array is developed to simultaneously detect TOX, RUB, CMV, HSV-I and HSV-II respectively with five functionalized beads. A compact liquid handling module has been developed to automate the sandwiched chemiluminescence immunoassay within the one-dimensional beads array of the microfluidic chip. A precise ram pump is adopted to not only add reagent into the microfluidic chip from outside, but also facilitate elaborate fluid control inside the microfluidic chip for improved performance. A large-size waste chamber with a liquid-absorbing sponge holds the waste reagent within the microfluidic chip to prevent backflow. The one-dimensional beads array is heated from double-sides at 37 ℃ for sensitive detection with reduced time. A sensitive CMOS camera is adopted to take chemiluminescence image from the one-dimensional beads array, and a custom processing algorithm is adopted to analyze the image. For each serum sample, five different infections can be simultaneously detected with the automated microfluidic system. Experimental results show that efficient, sensitive, and accurate multiplexed TORCH detection can be conveniently achieved with the integrated microfluidic system.</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-11-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4139633","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 protective effect of puerarin-loaded mesoporous silicon nanoparticles on alcoholic hepatitis through mTOR-mediated autophagy pathway","authors":"Xia-xia Zhang,&nbsp;Yan-fei Lang,&nbsp;Xin Li,&nbsp;Zheng Li,&nbsp;You-qing Xu,&nbsp;Hong-qian Chu","doi":"10.1007/s10544-022-00622-2","DOIUrl":"10.1007/s10544-022-00622-2","url":null,"abstract":"<div><p>Puerarin, a bioactive flavone compound isolated from Pueraria (Wild.), provides hepatoprotection by anti-inflammatory, anti-alcoholism, and regulating mechanistic target of rapamycin (mTOR). Building evidence suggests that the activation of mTOR reduces liver injuries associated with alcohol consumption and metabolism. However, the poor water solubility, low bioavailability, and short half-life of puerarin hinder its clinical application. The utility of mesoporous silicon nanoparticles (MSNs) can improve traditional Chinese medicine limitations. Stober methods were used to fabricate MSNs@Pue, and the size, zeta potentials and drug encapsulation efficiency were characterized by a series of analytical methods. IVIS Imaging System demonstrated liver-targeted bio-distribution, and then high-throughput sequencing, immunoproteomics and ultrastructure methods indicated autophagy related protective mechanism, followed by curative effect evaluation for the treatment efficacy. An acute-on chronic ethanol-drinking according to Gao-binge model induced alcoholic hepatitis (AH) pathology and resulted in hepatic hyper-autophagy, which was improved with MSNs@Pue administration (puerarin: 30 mM, 42 mg/kg; intravenously [i.v.]). Ethanol-fed mice were found to have increased expression of autophagy-related proteins (Atg3, Atg7, LC3 and p62). In contrast, MSNs@Pue administration significantly decreased the expression of these proteins and alleviated fatty droplets infiltration in damaged liver. Furthermore, acute-on-chronic ethanol feeding also resulted in the activiation of ERK activation and mTOR expression, which were reversed with MSNs@Pue administration and better than the usage of puerarin alone. Results point to MSNs@Pue mediated ERK/mTOR signaling pathway activation as a possible protective strategy to improve AH, which provides a strategy and evidence for treating liver disease using an MSN delivery system.\u0000</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10544-022-00622-2.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5131753","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":"Digital filtering dissemination for optimizing impedance cytometry signal quality and counting accuracy","authors":"Brandon K. Ashley,&nbsp;Umer Hassan","doi":"10.1007/s10544-022-00636-w","DOIUrl":"10.1007/s10544-022-00636-w","url":null,"abstract":"<div><p>Improving biosensor performance which utilize impedance cytometry is a highly interested research topic for many clinical and diagnostic settings. During development, a sensor’s design and external factors are rigorously optimized, but improvements in signal quality and interpretation are usually still necessary to produce a sensitive and accurate product. A common solution involves digital signal processing after sample analysis, but these methods frequently fall short in providing meaningful signal outcome changes. This shortcoming may arise from a lack of investigative research into selecting and using signal processing functions, as many choices in current sensors are based on either theoretical results or estimated hypotheses. While a ubiquitous condition set is improbable across diverse impedance cytometry designs, there lies a need for a streamlined and rapid analytical method for discovering those conditions for unique sensors. Herein, we present a comprehensive dissemination of digital filtering parameters applied on experimental impedance cytometry data for determining the limits of signal processing on signal quality improvements. Various filter orders, cutoff frequencies, and filter types are applied after data collection for highest achievable noise reduction. After designing and fabricating a microfluidic impedance cytometer, 9 µm polystyrene particles were measured under flow and signal quality improved by 6.09 dB when implementing digital filtering. This approached was then translated to isolated human neutrophils, where similarly, signal quality improved by 7.50 dB compared to its unfiltered original data. By sweeping all filtering conditions and devising a system to evaluate filtering performance both by signal quality and object counting accuracy, this may serve as a framework for future systems to determine their appropriately optimized filtering configuration.</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10544-022-00636-w.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5103201","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":"A comparative analysis of low intensity ultrasound effects on living cells: from simulation to experiments","authors":"Giulia Tamboia,&nbsp;Michele Campanini,&nbsp;Veronica Vighetto,&nbsp;Luisa Racca,&nbsp;Luca Spigarelli,&nbsp;Giancarlo Canavese,&nbsp;Valentina Cauda","doi":"10.1007/s10544-022-00635-x","DOIUrl":"10.1007/s10544-022-00635-x","url":null,"abstract":"<div><p>Ultrasounds are already broadly exploited in clinical diagnostics and are now becoming a powerful and not harmful tool in antitumoral therapies, as they are able to produce damages towards cancer cells, thank to inertial cavitation and temperature increase. The use of US alone or combined to molecular compounds, microbubbles or solid-state nanoparticles is the focus of current research and clinical trials, like thermoablation, drug sonoporation or sonodynamic therapies. In the present work, we discuss on the non-thermal effects of ultrasound and the conditions which enable oxygen radical production and which role they can have in provoking the death of different cancer cell lines. In this perspective, we set a mathematical model to predict the pressure spatial distribution in a defined water sample volume and thus obtain a map of acoustic pressures and acoustic intensities of the applied ultrasound at different input powers. We then validate and verify these numerical results with direct acoustic measurements and by detecting the production of reactive oxygen species (ROS) by means of sonochemiluminescence (SCL) and electron paramagnetic resonance (EPR) spectroscopy, applied to the same water sample volume and using the same US input parameters adopted in the simulation. Finally, the various US conditions are applied to two different set of cancer cell lines, a cervical adenocarcinoma and a hematological cancer, Burkitt’s lymphoma. We hypothesize how the ROS generation can influence the recorded cell death. In a second set of experiments, the role of semiconductor metal oxide nanocrystals, i.e. zinc oxide, is also evaluated by adding them to the water and biological systems. In particular, the role of ZnO in enhancing the ROS production is verified. Furthermore, the interplay among US and ZnO nanocrystals is evaluated in provoking cancer cell death at specific conditions. This study demonstrates a useful correlation between numerical simulation and experimental acoustic validation as well as with ROS measurement at both qualitative and quantitative levels during US irradiation of simple water solution. It further tries to translate the obtained results to justify one of the possible mechanisms responsible of cancer cell death. It thus aims to pave the way for the use of US in cancer therapy and a better understanding on the non-thermal effect that a specific set of US parameters can have on cancer cells cultured <i>in vitro</i>.</p><h3>Graphical abstract</h3>\u0000 <figure><div><div><div><picture><source><img></source></picture></div></div></div></figure>\u0000 </div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-10-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10544-022-00635-x.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4954916","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":"Compartmentalized organ-on-a-chip structure for spatiotemporal control of oxygen microenvironments","authors":"Kaisa Tornberg,&nbsp;Hannu Välimäki,&nbsp;Silmu Valaskivi,&nbsp;Antti-Juhana Mäki,&nbsp;Matias Jokinen,&nbsp;Joose Kreutzer,&nbsp;Pasi Kallio","doi":"10.1007/s10544-022-00634-y","DOIUrl":"10.1007/s10544-022-00634-y","url":null,"abstract":"<div><p>Hypoxia is a condition where tissue oxygen levels fall below normal levels. In locally induced hypoxia due to blood vessel blockage, oxygen delivery becomes compromised. The site where blood flow is diminished the most forms a zero-oxygen core, and different oxygenation zones form around this core with varying oxygen concentrations. Naturally, these differing oxygen microenvironments drive cells to respond according to their oxygenation status. To study these cellular processes in laboratory settings, the cellular gas microenvironments should be controlled rapidly and precisely. In this study, we propose an organ-on-a-chip device that provides control over the oxygen environments in three separate compartments as well as the possibility of rapidly changing the corresponding oxygen concentrations. The proposed device includes a microfluidic channel structure with three separate arrays of narrow microchannels that guide gas mixtures with desired oxygen concentrations to diffuse through a thin gas-permeable membrane into cell culture areas. The proposed microfluidic channel structure is characterized using a 2D ratiometric oxygen imaging system, and the measurements confirm that the oxygen concentrations at the cell culture surface can be modulated in a few minutes. The structure is capable of creating hypoxic oxygen tension, and distinct oxygen environments can be generated simultaneously in the three compartments. By combining the microfluidic channel structure with an open-well coculture device, multicellular cultures can be established together with compartmentalized oxygen environment modulation. We demonstrate that the proposed compartmentalized organ-on-a-chip structure is suitable for cell culture.</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-10-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10544-022-00634-y.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4836359","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":"A multi-material platform for imaging of single cell-cell junctions under tensile load fabricated with two-photon polymerization","authors":"Jordan Rosenbohm,&nbsp;Grayson Minnick,&nbsp;Bahareh Tajvidi Safa,&nbsp;Amir Monemian Esfahani,&nbsp;Xiaowei Jin,&nbsp;Haiwei Zhai,&nbsp;Nickolay V. Lavrik,&nbsp;Ruiguo Yang","doi":"10.1007/s10544-022-00633-z","DOIUrl":"10.1007/s10544-022-00633-z","url":null,"abstract":"<div><p>We previously reported a single-cell adhesion micro tensile tester (SCAμTT) fabricated from IP-S photoresin with two-photon polymerization (TPP) for investigating the mechanics of a single cell-cell junction under defined tensile loading. A major limitation of the platform is the autofluorescence of IP-S, the photoresin for TPP fabrication, which significantly increases background signal and makes fluorescent imaging of stretched cells difficult. In this study, we report the design and fabrication of a new SCAμTT platform that mitigates autofluorescence and demonstrate its capability in imaging a single cell pair as its mutual junction is stretched. By employing a two-material design using IP-S and IP-Visio, a photoresin with reduced autofluorescence, we show a significant reduction in autofluorescence of the platform. Further, by integrating apertures onto the substrate with a gold coating, the influence of autofluorescence on imaging is almost completely mitigated. With this new platform, we demonstrate the ability to image a pair of epithelial cells as they are stretched up to 250% strain, allowing us to observe junction rupture and F-actin retraction while simultaneously recording the accumulation of over 800 kPa of stress in the junction. The platform and methodology presented here can potentially enable detailed investigation of the mechanics of and mechanotransduction in cell-cell junctions and improve the design of other TPP platforms in mechanobiology applications.</p><h3>Graphical abstract</h3>\u0000 <figure><div><div><div><picture><source><img></source></picture></div></div></div></figure>\u0000 </div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10544-022-00633-z.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4363945","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":"Biosensors for detection of prostate cancer: a review","authors":"Sourav Sarkar,&nbsp;Manashjit Gogoi,&nbsp;Mrityunjoy Mahato,&nbsp;Abhijeet Balwantrao Joshi,&nbsp;Arup Jyoti Baruah,&nbsp;Prashant Kodgire,&nbsp;Polina Boruah","doi":"10.1007/s10544-022-00631-1","DOIUrl":"10.1007/s10544-022-00631-1","url":null,"abstract":"<div><p>Diagnosis of prostate cancer (PC) has posed a challenge worldwide due to the sophisticated and costly diagnostics tools, which include DRE, TRUS, GSU, PET/CT scan, MRI, and biopsy. These diagnostic techniques are very helpful in the detection of PCs; however, all the techniques have their serious limitations. Biosensors are easier to fabricate and do not require any cutting-edge technology as required for other imaging techniques. In this regard, point-of-care (POC) biosensors are important due to their portability, convenience, low cost, and fast procedure. This review explains the various existing diagnostic tools for the detection of PCs and the limitation of these methods. It also focuses on the recent studies on biosensors technologies as an alternative to the conventional diagnostic techniques for the detection of PCs.\u0000</p><h3>Graphical abstract</h3>\u0000 <figure><div><div><div><picture><source><img></source></picture></div></div></div></figure>\u0000 </div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s10544-022-00631-1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"5099929","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":"A flexible implantable microelectrode array for recording electrocorticography signals from rodents","authors":"Suman Chatterjee,&nbsp;Tushar Sakorikar,&nbsp;Arjun BS,&nbsp;Rathin K. Joshi,&nbsp;Abhay Sikaria,&nbsp;Mahesh Jayachandra,&nbsp;Vikas V,&nbsp;Hardik J. Pandya","doi":"10.1007/s10544-022-00632-0","DOIUrl":"10.1007/s10544-022-00632-0","url":null,"abstract":"<div><p>Electrocorticography signals, the intracranial recording of electrical signatures of the brain, are recorded by non-penetrating planar electrode arrays placed on the cortical surface. Flexible electrode arrays minimize the tissue damage upon implantation. This work shows the design and development of a 32-channel flexible microelectrode array to record electrocorticography signals from the rat's brain. The array was fabricated on a biocompatible flexible polyimide substrate. A titanium/gold layer was patterned as electrodes, and a thin polyimide layer was used for insulation. The fabricated microelectrode array was mounted on the exposed somatosensory cortex of the right hemisphere of a rat after craniotomy and incision of the dura. The signals were recorded using OpenBCI Cyton Daisy Biosensing Boards. The array faithfully recorded the baseline electrocorticography signals, the induced epileptic activities after applying a convulsant, and the recovered baseline signals after applying an antiepileptic drug. The signals recorded by such fabricated microelectrode array from anesthetized rats demonstrate its potential to monitor electrical signatures corresponding to epilepsy. Finally, the time–frequency analyses highlight the difference in spatiotemporal features of baseline and evoked epileptic discharges.\u0000</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 4","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"4704586","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":"Dielectrophoretic separation of blood cells","authors":"Maria E. P. Emmerich,&nbsp;Anne-Sophie Sinnigen,&nbsp;Peter Neubauer,&nbsp;Mario Birkholz","doi":"10.1007/s10544-022-00623-1","DOIUrl":"10.1007/s10544-022-00623-1","url":null,"abstract":"<div><p>Microfluidic dielectrophoretic (DEP) devices enable the label-free separation and isolation of cells based on differences in their electrophysiological properties. The technique can serve as a tool in clinical diagnostics and medical research as it facilitates the analysis of patient-specific blood composition and the detection and isolation of pathogenic cells like circulating tumor cells or malaria-infected erythrocytes. This review compares different microfluidic DEP devices to separate platelets, erythrocytes and leukocytes including their cellular subclasses. An overview and experimental setups of different microfluidic DEP devices for the separation, trapping and isolation or purification of blood cells are detailed with respect to their technical design, electrode configuration, sample preparation, applied voltage and frequency and created DEP field based and related to the separation efficiency. The technique holds the promise that results can quickly be attained in clinical and ambulant settings. In particular, point-of-care-testing scenarios are favored by the extensive miniaturization, which would be enabled by microelectronical integration of DEP devices.\u0000</p></div>","PeriodicalId":490,"journal":{"name":"Biomedical Microdevices","volume":"24 3","pages":""},"PeriodicalIF":2.8,"publicationDate":"2022-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9411249/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40415735","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}