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The development of affordable, reliable, and rapid diagnostic devices is crucial for monitoring immunological responses using a drop of blood. However, conventional automated diagnostic devices typically involve expensive and impractical robotic fluid-handling approaches. Herein, we developed an integrated cell analyzer comprising a cylindrical sample cartridge connected to a direct current motor and a compact fluorescence imaging module. Sample mixing and loading are performed automatically by a programmable sequence of single motor rotation controlled by an Android application. Two distinct stained immune cell samples can be identified by using two types of fluorescence imaging modes. The effectiveness of mixing performance in antigen-antibody (Ag-Ab) reactions was assessed through a compound objective lens that collects weak fluorescence emitted by the cell membrane. Active mixing with bidirectional rotation of the cartridge in a confined space shortened the Ag-Ab reaction time by a factor of 3.3 and achieved cell counting with higher accuracy while reducing reagent consumption by 4 times compared to the conventional incubation method. High-intensity fluorescence images of cells labeled with a nucleic acid stain were acquired through a single-lens-based fluorescence imaging module with a large field of view (FOV) in an unconventional detection chamber with a curved substrate. Compared with a flat chamber, the curved detection chamber reduces the effects of field curvature and provides aberration-free wide-FOV images, even with a simple lens. Our integrated cell analyzer thus offers a practical and cost-effective solution for monitoring patient immune responses in point-of-care settings.
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Sistemas Automatizados de Assistência Junto ao Leito , Humanos , FluorescênciaRESUMO
Computational techniques can enhance personalized hyperthermia-treatment planning by calculating tissue energy absorption and temperature distribution. This study determined the effect of tumor properties on energy absorption, temperature mapping, and thermal dose distribution in mild radiofrequency hyperthermia using a mouse xenograft model. We used a capacitive-heating radiofrequency hyperthermia system with an operating frequency of 13.56â¯MHz for in vivo mouse experiments and performed simulations on a computed tomography mouse model. Additionally, we measured the dielectric properties of the tumors and considered temperature dependence for thermal properties, metabolic heat generation, and perfusion. Our results showed that dielectric property variations were more dominant than thermal properties and other parameters, and that the measured dielectric properties provided improved temperature-mapping results relative to the property values taken from previous study. Furthermore, consideration of temperature dependency in the bio heat-transfer model allowed elucidation of precise thermal-dose calculations. These results suggested that this method might contribute to effective thermoradiotherapy planning in clinics.
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Hipertermia Induzida/métodos , Neoplasias/química , Neoplasias/radioterapia , Termografia , Animais , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/radioterapia , Simulação por Computador , Xenoenxertos , Humanos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/radioterapia , Camundongos , Modelos Biológicos , Ondas de Rádio , TemperaturaRESUMO
Collective bacterial dynamics plays a crucial role in colony development. Although many research groups have studied the behavior of fluidic swarm colonies, the detailed mechanics of its motion remains elusive. Here, we developed a visualization method using submicron fluorescent beads for investigating the flow field in a thin layer of fluid that covers a Bacillus subtilis swarm colony growing on an agar plate. The beads were initially embedded in the agar plate and subsequently distributed spontaneously at the upper surface of the expanding colony. We conducted long-term live cell imaging of the B. subtilis colony using the fluorescent tracers, and obtained high-resolution velocity maps of microscale vortices in the swarm colony using particle image velocimetry. A distinct periodic fluctuation in the average speed and vorticity of flow in swarm colony was observed at the inner region of the colony, and correlated with the switch between bacterial swarming and growth phases. At the advancing edge of the colony, both the magnitudes of velocity and vorticity of flow in swarm colony were inversely correlated with the spreading speed of the swarm edge. The advanced imaging tool developed in this study would facilitate further understanding of the effect of micro vortices in swarm colony on the collective dynamics of bacteria.
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Ágar , Bacillus subtilis/crescimento & desenvolvimento , Meios de CulturaRESUMO
Phase contrast microscopy (PCM) is a widely used analytical method for airborne asbestos, but it is unable to distinguish asbestos from non-asbestos fibers and requires time-consuming and laborious manual counting of fibers. Previously, we developed a high-throughput microscopy (HTM) method that could greatly reduce human intervention and analysis time through automated image acquisition and counting of fibers. In this study, we designed a dual-mode HTM (DM-HTM) device for the combined reflection and fluorescence imaging of asbestos, and automated a series of built-in image processing commands of ImageJ software to test its capabilities. We used DksA, a chrysotile-adhesive protein, for selective detection of chrysotile fibers in the mixed dust-free suspension of crysotile and amosite prepared in the laboratory. We demonstrate that fluorescently-stained chrysotile and total fibers can be identified and enumerated automatically in a high-throughput manner by the DM-HTM system. Combined with more advanced software that can correctly identify overlapping and branching fibers and distinguish between fibers and elongated dust particles, the DM-HTM method should enable fully automated counting of airborne asbestos.
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Electrospun nanofibrous structures provide good performance to scaffolds in tissue engineering. We measured the local diffusion coefficients of 3-kDa FITC-dextran in line patterns of electrospun nanofibrous structures fabricated by the direct-write electrospinning (DWES) technique using the fluorescence recovery after photobleaching (FRAP) method. No significant differences were detected between DWES line patterns fabricated with polymer supplied at flow rates of 0.1 and 0.5 mL/h. The oxygen diffusion coefficients of samples were estimated to be ~92%-94% of the oxygen diffusion coefficient in water based on the measured diffusion coefficient of 3-kDa FITC-dextran. We also simulated cell growth and distribution within spatially patterned scaffolds with struts consisting of either oxygen-permeable or non-permeable material. The permeable strut scaffolds exhibited enhanced cell growth. Saturated depths at which cells could grow to confluence were 15% deeper for the permeable strut scaffolds than for the non-permeable strut scaffold.
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Nanofibras/química , Engenharia Tecidual/métodos , Contagem de Células , Proliferação de Células , Dextranos/química , Difusão , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Oxigênio/metabolismoRESUMO
Integrated wearable bioelectronic health monitoring systems have revealed new possibilities for collecting personalized physiological information. Wearable sweat sensors have the potential to noninvasively measure valuable biomarkers. Mapping sweat and skin-temperature throughout the body can provide detailed information on the human body. However, existing wearable systems cannot evaluate such data. Here, we report a multifunctional wearable platform that can wirelessly measure local sweat loss, sweat chloride concentration, and skin temperature. The approach combines a reusable electronics module to monitor skin temperature and a microfluidic module for monitoring sweat loss and sweat chloride concentration. The miniaturized electronic system takes temperature measurements from the skin and wirelessly transmits the obtained data to a user device using Bluetooth technology. In contrast, the microfluidic system provides accurate colorimetric analysis of the chloride concentration and sweat loss. Thus, this integrated wearable system has great application potential in individualized health management systems for sports researchers and competitors and can also be applied in clinical settings.
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Suor , Dispositivos Eletrônicos Vestíveis , Humanos , Temperatura Corporal , Cloretos , PeleRESUMO
BACKGROUND: Needle-free jet injection (NFJI) systems enable a controlled and targeted delivery of drugs into skin tissue. However, a scarce understanding of their underlying mechanisms has been a major deterrent to the development of an efficient system. Primarily, the lack of a suitable visualization technique that could capture the dynamics of the injected fluid-tissue interaction with a microsecond range temporal resolution has emerged as a main limitation. A conventional needle-free injection system may inject the fluids within a few milliseconds and may need a temporal resolution in the microsecond range for obtaining the required images. However, the presently available imaging techniques for skin tissue visualization fail to achieve these required spatial and temporal resolutions. Previous studies on injected fluid-tissue interaction dynamics were conducted using in vitro media with a stiffness similar to that of skin tissue. However, these media are poor substitutes for real skin tissue, and the need for an imaging technique having ex vivo or in vivo imaging capability has been echoed in the previous reports. METHODS: A near-infrared imaging technique that utilizes the optical absorption and fluorescence emission of indocyanine green dye, coupled with a tissue clearing technique, was developed for visualizing a NFJI in an ex vivo porcine skin tissue. RESULTS: The optimal imaging conditions obtained by considering the optical properties of the developed system and mechanical properties of the cleared ex vivo samples are presented. Crucial information on the dynamic interaction of the injected liquid jet with the ex vivo skin tissue layers and their interfaces could be obtained. CONCLUSIONS: The reported technique can be instrumental for understanding the injection mechanism and for the development of an efficient transdermal NFJI system as well.
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Injections into or through the skin are common drug or vaccine administration routes, which can be achieved with conventional needles, microneedles, or needle-free jet injections (NFJI). Understanding the transport mechanism of these injected fluids is critical for the development of effective drug administration devices. NFJI devices are distinct from traditional injection techniques by their route and time scale, which relies on a propelled microjet with sufficient energy to penetrate the skin surface and deliver the drug into the targeted region. The injected fluid interacts with multiple skin tissue layers and interfaces, which implies that the corresponding injection profile is dependent on their mechanical properties. In this study, we address the lack of fundamental knowledge on the impact of these interfaces on the injection profiles of NFJI devices.
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Sistemas de Liberação de Medicamentos , Pele , Sistemas de Liberação de Medicamentos/métodos , Desenho de Equipamento , Injeções a Jato/métodos , Preparações FarmacêuticasRESUMO
Wearable sensors and machine learning algorithms are widely used for predicting an individual's thermal sensation. However, most of the studies are limited to controlled laboratory experiments with inconvenient wearable sensors without considering the dynamic behavior of ambient conditions. In this study, we focused on predicting individual dynamic thermal sensation based on physiological and psychological data. We designed a smart face mask that can measure skin temperature (SKT) and exhaled breath temperature (EBT) and is powered by a rechargeable battery. Real-time human experiments were performed in a subway cabin with twenty male students under natural conditions. The data were collected using a smartphone application, and we created features using the wavelet decomposition technique. The bagged tree algorithm was selected to train the individual model, which showed an overall accuracy and f-1 score of 98.14% and 96.33%, respectively. An individual's thermal sensation was significantly correlated with SKT, EBT, and associated features.
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Máscaras , Ferrovias , Humanos , Temperatura Cutânea , Temperatura , Sensação Térmica/fisiologiaRESUMO
Mechanical forces from blood flow and pressure (hemodynamic forces) contribute to the formation and shaping of the blood vascular network during embryonic development. Previous studies have demonstrated that hemodynamic forces regulate signaling and gene expression in endothelial cells that line the inner surface of vascular tubes, thereby modifying their cellular state and behavior. Given its important role in vascular development, we still know very little about the quantitative aspects of hemodynamics that endothelial cells experience due to the difficulty in measuring forces in vivo. In this study, we sought to determine the magnitude of wall shear stress (WSS) exerted on ECs by blood flow in different vessel types and how it evolves during development. Utilizing the zebrafish as a vertebrate model system, we have established a semi-automated high-throughput fluorescent imaging system to capture the flow of red blood cells in an entire zebrafish between 2- and 6-day post-fertilization (dpf). This system is capable of imaging up to 50 zebrafish at a time. A semi-automated analysis method was developed to calculate WSS in zebrafish trunk vessels. This was achieved by measuring red blood cell flow using particle tracking velocimetry analysis, generating a custom-made script to measure lumen diameter, and measuring local tube hematocrit levels to calculate the effective blood viscosity at each developmental stage. With this methodology, we were able to determine WSS magnitude in different vessels at different stages of embryonic and larvae growth and identified developmental changes in WSS, with absolute levels of peak WSS in all vessel types falling to levels below 0.3 Pa at 6 dpf. Additionally, we discovered that zebrafish display an anterior-to-posterior trend in WSS at each developmental stage.
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Inhalation of airborne asbestos causes serious health problems such as lung cancer and malignant mesothelioma. The phase-contrast microscopy (PCM) method has been widely used for estimating airborne asbestos concentrations because it does not require complicated processes or high-priced equipment. However, the PCM method is time-consuming and laborious as it is manually performed off-site by an expert. We have developed a high-throughput microscopy (HTM) method that can detect fibers distinguishable from other spherical particles in a sample slide by image processing both automatically and quantitatively. A set of parameters for processing and analysis of asbestos fiber images was adjusted for standard asbestos samples with known concentrations. We analyzed sample slides containing airborne asbestos fibers collected at 11 different workplaces following PCM and HTM methods, and found a reasonably good agreement in the asbestos concentration. Image acquisition synchronized with the movement of the robotic sample stages followed by an automated batch processing of a stack of sample images enabled us to count asbestos fibers with greatly reduced time and labors. HTM should be a potential alternative to conventional PCM, moving a step closer to realization of on-site monitoring of asbestos fibers in air.
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Amianto/análise , Ensaios de Triagem em Larga Escala/métodos , Microscopia de Contraste de Fase/métodos , Material Particulado/análise , Processamento de Imagem Assistida por Computador/métodosRESUMO
SIGNIFICANCE: Dry or moist skin-contact thermal stimulation for vein puncture (VP) and vein cannulation (VC) may not be feasible for sensitive skin. For a damaged, burned, or dark skin, near-infrared (NIR) imaging is preferred to visualize a vein. Postprocessing of NIR images is always required because the skin is a reflective material and veins need segmentation for quantitative analysis. AIM: Our pilot study aims to observe the effect of noncontact local heating on the superficial metacarpal veins in the dorsal surface of the hand and to visualize vein dynamics using an NIR imaging system. APPROACH: Our experiment consists of studies A and B at two ambient temperatures, 19°C and 25°C. A simple reflection-based NIR imaging system was installed to acquire sequential vein images for 5 min before and after applying 10 min of radiant thermal stimulation. To measure the vein diameter (VD), we trained a convolutional neural network (CNN) on sequential raw images to predict vein-segmentation masks as output images. Later these masked images were postprocessed for the VD measurements. RESULTS: The average VD was significantly increased after thermal stimulation in study A. The maximum increments in VD were 39.3% and 9.19%, 1 min after thermal stimulation in studies A and B, respectively. Both the VD and skin temperature (Tskin) follow negative exponentials in time, and the VD is proportional to Tskin. A multiple linear-regression model was made to predict the final VD. A significant difference was observed in the change of the VD. CONCLUSIONS: NIR imaging with CNN can be used for quantitative analyses of vein dynamics. This finding can be further extended to develop real-time, image-guided medical devices by integrating them with a radiant heater and to assist medical practitioners in achieving high success rates for VP or VC.
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Diagnóstico por Imagem , Veias , Redes Neurais de Computação , Projetos Piloto , Temperatura , Veias/diagnóstico por imagemRESUMO
Core body temperature (Tcore ) is a key indicator of personal thermal comfort and serves as a monitor of thermal strain. Multi-parametric sensors are not practical for estimating core temperature because they require long data collection times and a wide variety of settings. This study introduces dorsal hand vein dynamics as a novel indicator along with heart rate (HR) and dorsal hand skin temperature (Thand ) for predicting Tcore during rest following Tcore elevation. Twelve healthy males aged 27 ± 9 years old participated in the experiment. The experimental procedure consisted of a 10-min rest followed by 60 min of passive heat stress induced by leg immersion in hot water at 42°C and a 40-min thermal relaxation period after the legs were removed from the water. A near-infrared (NIR) imaging system was configured to monitor the dorsal hand veins during the entire experimental session. The values of HR, Thand , and Tcore were continuously monitored while the ambient temperature and relative humidity (RH) were maintained in a climate chamber at 20°C and 50%, respectively. Our selected predictor parameters demonstrated similar patterns in the Tcore such that the value increased as a result of passive heat stress and decreased in the thermal relaxation phase. The experimental data were divided into two phases: thermal stress and relaxation. At the resting condition, inclusion of the hand vein diameter (VD) improved the multiple linear regression value (R2 ) about 26%. At the relaxation phase, however, training regressions R2 = 0.68 and R2 = 0.94 were observed in the regression model with and without considering VD, respectively. The test regression value of R2 = 0.88 and the root mean square error (RMSE) of 0.18°C showed good agreement with the predicted values. These findings demonstrate acceptable validity of the non-invasive Tcore estimation at the resting condition. In particular, the inclusion of VD as a predictor in the regression analysis increases the prediction accuracy with a lower RMSE value.
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We developed microfluidic-based pure chitosan microfibers (approximately 1 meter long, 70-150 microm diameter) for liver tissue engineering applications. Despite the potential of the chitosan for creating bio-artificial liver chips, its major limitation is the inability to fabricate pure chitosan-based microstructures with controlled shapes because of the mechanical weakness of the pure chitosan. Previous studies have shown that chitosan micro/nanofibers can be fabricated by using chemicals and electrospinning techniques. However, there is no paper regarding pure chitosan-based microfibers in a microfluidic device. This paper suggests a unique method to fabricate pure chitosan microfibers without any chemical additive. We also analyzed the chemical, mechanical, and diffusion properties of pure chitosan microfibers. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrometry and electron spectroscopy for chemical analysis (ESCA) were used to analyze the chemical composition of the synthesized chitosan microfibers. We measured the mechanical axial-force and diffusion coefficient in pure chitosan-based microfibers using fluorescence recovery after photobleaching (FRAP) techniques. Furthermore, to evaluate the capability of the microfibers for liver tissue formation, hepatoma HepG2 cells were seeded onto the chitosan microfibers. The functionality of these hepatic cells cultured on chitosan microfibers was analyzed by measuring albumin secretion and urea synthesis. Therefore, this pure chitosan-based microfiber chip could be a potentially useful method for liver tissue engineering applications.
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Quitosana/síntese química , Fígado Artificial , Fígado/citologia , Microfluídica , Engenharia Tecidual/instrumentação , Alicerces Teciduais/química , Albuminas/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Quitosana/química , Quitosana/farmacologia , Difusão , Dimetilpolisiloxanos/química , Recuperação de Fluorescência Após Fotodegradação , Humanos , Fígado/metabolismo , Fígado/fisiologia , Teste de Materiais , Microscopia de Fluorescência , Engenharia Tecidual/métodos , Ureia/metabolismoRESUMO
It is reported that a conversion from mycophenolate mofetil (MMF) to enteric-coated mycophenolate sodium (EC-MPS) relieves gastrointestinal (GI) symptom burden and improves health-related quality of life (HRQoL). However, it is unclear whether renal transplant recipients using tacrolimus receive the same benefit from the conversion. In this prospective, multi-center, open-label trial, patients were categorized into two groups by their GI symptom screening. Equimolar EC-MPS (n=175) was prescribed for patients with GI burdens; those with no complaints remained on MMF (n=83). Gastrointestinal Symptom Rating Scale (GSRS) and Gastrointestinal Quality of Life Index (GIQLI) were evaluated at baseline and after one month. Patients and physicians completed Overall Treatment Effect (OTE) at one month. EC-MPS-converted patients had worse GSRS and GIQLI scores at baseline than MMF-continued patients (all P<0.001). Significant improvements in GSRS and GIQLI scores were observed for EC-MPS-converted patients at one month, but MMF-continued patients showed worsened GSRS scores (all P<0.05). OTE scale indicated that EC-MPS patients improved in overall GI symptoms and HRQoL more than MMF patients did (P<0.001). In tacrolimus-treated renal transplant recipients with GI burdens, a conversion from MMF to EC-MPS improves GI-related symptoms and HRQoL.
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Gastroenteropatias/induzido quimicamente , Imunossupressores/efeitos adversos , Transplante de Rim , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/efeitos adversos , Adolescente , Adulto , Idoso , Feminino , Rejeição de Enxerto/tratamento farmacológico , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/uso terapêutico , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/administração & dosagem , Ácido Micofenólico/uso terapêutico , Qualidade de Vida , Inquéritos e Questionários , Comprimidos com Revestimento Entérico , Tacrolimo/uso terapêuticoRESUMO
Needle-free jet injectors are non-invasive systems having intradermal drug delivery capabilities. At present, they revolutionize the next phase of drug delivery and therapeutic applications in the medical industry. An efficiently designed injection chamber can reduce the energy consumption required to achieve the maximum penetration depth in skin tissue. In this study, the authors explored the effect of various geometrical parameters using a computational fluid dynamics tool. Peak stagnation pressure during the initial phase of the injection procedure was considered as the quantifier for comparison because of its proportional relationship with the initial penetration depth during the injection process. Peak stagnation pressure indicates the maximum energy transformation that could happen between the microjet and skin tissues for an injection procedure. The results of this study indicated a tradeoff that exists between the attainable density and velocity of the microjet on the skin surface with variation in nozzle diameter; the optimum nozzle diameter was found to be within 200-250 µm under the present conditions. The authors also observed a discrepancy in the peak stagnation pressure value for lower filling ratios with variation in chamber diameter; hence, filling ratio of at least 50% was recommended for such systems. Furthermore, a 150% increase in the peak stagnation pressure was obtained with an angle of entry of 10°. In general, this study could provide valuable insights into the effect of geometrical parameters in the fluid dynamics characteristics of propelled microjets from the nozzle of a needle-free jet injector. Such information could be useful for the design of a mechanically driven needle-free jet injector having limited control over the energizing mechanism.
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Hidrodinâmica , Pele , Sistemas de Liberação de Medicamentos , Desenho de Equipamento , Injeções a JatoRESUMO
A nut-and-bolt microfluidic system was previously developed for a point-of-care (POC) human immunodeficiency virus (HIV) test and was able to acquire images of CD4 (cluster of differentiation 4) + T-lymphocytes in a sample drop of blood followed by image analysis. However, as the system was not fully integrated with a sample reaction module, the mixing of the sample with the antibody reagent was carried out manually. To achieve a rapid reaction with a reduced amount of costly reagent in a POC diagnostic system, an efficient sample mixing function must be implemented. Here, we propose a novel method to drastically accelerate the process of sample mixing and increase the reaction rate in the nut-and-bolt microfluidic system, where the sample is mixed with the reagent in a reaction chamber formed by connecting a nut with a bolt-like sample cartridge. The mixing is facilitated by rotating the sample cartridge bidirectionally using a DC motor, which agitates the sample in a chaotic manner. A microbead complex formed by the avidin-biotin interaction was used as a model reaction system to examine the feasibility of our mixing module. We found that the reaction time for the avidin-biotin binding by mixing was 7.5 times shorter than in the incubation method, achieving a reaction efficiency of over 95%. The performance of our mixing system was further demonstrated by measuring the concentration of CD4 cells labeled with a fluorescent antibody in the blood sample. The antigen-antibody reaction mixing was faster by a factor of 20, reaching a reaction efficiency comparable to the conventional incubation method.
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The CD4 (cluster of differentiation 4) counting method is used to measure the number of CD4+ T-lymphocytes per microliter of blood and to evaluate the timing of the initiation of antiretroviral therapy as well as the effectiveness of treatment in patients with human immunodeficiency virus. We developed a three-dimensional helical minichannel-based sample cartridge in which a thread-like microgroove formed in the cylindrical surface and configured a particle-positioning and imaging system equipped with a single DC (direct current) motor that can be controlled by a smartphone application. Confinement and enrichment of CD4 cells within a sharp focal depth along the helical minichannel is accomplished by spinning the cylindrical sample cartridge at high speed before acquiring cell images and thus CD4+ cells with weak fluorescence intensity can be detected even in a channel much deeper than existing two-dimensional flat chambers without an autofocusing module. By detecting more cells in a larger sample volume, the accuracy of the CD4 cell count is improved by a factor of 5.8 with a channel of 500 µm depth and the precision is enhanced by a factor of 1.5 with a coefficient of variation of 2.6%.
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BACKGROUND/AIMS: Blood flow profiles in fiber bundles depend on the design of the arterial port and affects the biocompatibility of the hemodialyzer. We analyzed the effects of arterial port design on blood flow distribution in fiber bundles using nonintrusive imaging techniques. METHODS: The velocity fields in arterial ports and the hemodynamics in fiber bundles were analyzed for hemodialyzers with different configurations using particle image velocimetry and perfusion computed tomography. RESULTS: In a hemodialyzer with standard arterial ports, high blood flow profiles in the central and peripheral regions and low blood profiles in the middle region were developed due to jet flow and vortices around the jet. In a hemodialyzer with spiral arterial ports, higher flow profiles were developed due to the central vortices that decrease perfusion into the fiber bundles. CONCLUSION: The arterial port design of hemodialyzers should be optimized such that jet flow and vortices do not impair dialysis efficiency and biocompatibility.
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Diálise Renal/instrumentação , Artérias , Velocidade do Fluxo Sanguíneo , Humanos , Diálise Renal/métodosRESUMO
Mutations in cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel, cause cystic fibrosis. To investigate interactions of CFTR in living cells, we measured the diffusion of quantum dot-labeled CFTR molecules by single particle tracking. In multiple cell lines, including airway epithelia, CFTR diffused little in the plasma membrane, generally not moving beyond 100-200 nm. However, CFTR became mobile over micrometer distances after 1) truncations of the carboxy terminus, which contains a C-terminal PDZ (PSD95/Dlg/ZO-1) binding motif; 2) blocking PDZ binding by C-terminal green fluorescent protein fusion; 3) disrupting CFTR association with actin by expression of a mutant EBP50/NHERF1 lacking its ezrin binding domain; or 4) skeletal disruption by latrunculin. CFTR also became mobile when the cytoskeletal adaptor protein binding capacity was saturated by overexpressing CFTR or its C terminus. Our data demonstrate remarkable and previously unrecognized immobilization of CFTR in the plasma membrane and provide direct evidence that C-terminal coupling to the actin skeleton via EBP50/ezrin is responsible for its immobility.