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1.
IEEE Trans Biomed Circuits Syst ; 14(4): 705-714, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32746345

RESUMO

There is an unmet need for a low-cost instrumented technology for detecting sanitation-related malodor as an alert for maintenance around shared toilets and emerging technologies for onsite waste treatment. In this article, our approach to an electronic nose for sanitation-related malodor is based on the use of electrochemical gas sensors, and machine-learning techniques for sensor selection and odor classification. We screened 10 sensors from different vendors with specific target gases and recorded their response to malodor from fecal specimens and urine specimens, and confounding good odors such as popcorn. The analysis of 180 odor exposures data by two feature-selection methods based on mutual information indicates that, for malodor detection, the decision tree (DT) classifier with seven features from four sensors provides 88.0% balanced accuracy and 85.1% macro F1 score. However, the k-nearest-neighbors (KNN) classifier with only three features (from two sensors) obtains 83.3% balanced accuracy and 81.3% macro F1 score. For classification of urine against feces malodor, a balanced accuracy of 94.0% and a macro F1 score of 92.9% are achieved using only four features from three sensors and a logistic regression (LR) classifier.

2.
Water Environ Res ; 2020 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-32516457

RESUMO

Electrolysis of blackwater for disinfection and nutrient removal is a portable and scalable technology that can lessen the need for cities to construct large-scale wastewater treatment infrastructure and enable the safe onsite reuse of blackwater. Several systems for treating wastewater from single toilets are described in the literature, but there are few examples of systems designed to use electrolysis to treat blackwater from nearby toilets, which is a situation more common in densely packed urban living environments. In order to scale a single toilet electrolysis system to one that could service multiple toilets, computational fluid dynamic analysis was used to optimize the electrochemical reactor design, and laboratory and field-testing were used to confirm results. Design efforts included optimization of the reactor shape and mixing to improve treatment efficiency, as well as automated cleaning and salt injection to reduce maintenance and service requirements. PRACTITIONER POINTS: Design of a reverse polarity mechanism to enable in situ electrode cleaning and improve long-term electrode performance. Optimization of a hopper design and drainpipe location to collect and remove flaking precipitates and mitigate maintenance issues. Design of an automated salt injection system to guarantee sufficient chloride levels for producing adequate chlorine residuals for consistent disinfection.

3.
Sci Total Environ ; 730: 138957, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32402964

RESUMO

A challenge in water reuse for toilet flushing in India and other Asian countries derives from pour flushing practices. It is a common assumption that the amount of pour flushed water used for personal cleansing is small in comparison to the cistern flush volume, however there is a knowledge gap regarding the actual contribution of each water source to the blackwater amount. In this study, digital water meters were used to measure the fraction of water from personal wash tap relative to cistern water that is used for toilet flushing. High temporal resolution measurements were carried in three different urban sites in the city of Coimbatore in the southern Indian state of Tamil Nadu where onsite sanitation treatment prototypes that may provide reclaimed water for cistern flushing are being tested. Data collected over periods of up to 2 months show that the contribution of the cistern flush to the total blackwater volume is low (14-40%). These data highlight an important factor to inform interventions designed around water reuse for flushing in world geographies where personal toilet cleansing by water is the common practice.

4.
J Head Trauma Rehabil ; 35(5): E422-E428, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32472839

RESUMO

OBJECTIVE: To determine the feasibility of short-term cardiovascular responses to postural change as a screening tool for mild traumatic brain injury (mTBI), using heart rate metrics that can be measured with a wearable electrocardiogram sensor. SETTING: Military TBI clinic. DESIGN: Data collected from active-duty service members who had sustained a medically diagnosed mTBI within the prior 72 hours and from age- and sex-matched controls. Cardiac data collected while participants performed a sequence of postural changes. MAIN MEASURES: Model classification compared with clinical mTBI diagnosis. RESULTS: Cardiac biomarkers of mTBI were identified and logistic regression classifiers for mTBI were developed from different subsets of biomarkers. The best model achieved 90% sensitivity and 69% specificity using data from 2 different postural changes. CONCLUSION: Noninvasive measurement of cardiovascular response to postural change is a promising approach for field-deployable post-mTBI screening.

5.
Sensors (Basel) ; 20(10)2020 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-32443709

RESUMO

Technology innovation in sanitation is needed for the 4.2 billion people worldwide, lacking safely managed sanitation services. A major requirement for the adoption of these technologies is the management of malodor around toilet and treatment systems. There is an unmet need for a low-cost instrumented technology for detecting the onset of sanitation malodor and triggering corrective actions. This study combines sensory data with low-cost gas sensor data on malodor emanating from feces. The response of 10 commercial electrochemical gas sensors was collected alongside olfactometric measurements. Odor from fecal specimens at different relevant dilution as well as specimens with pleasant odors as a control were evaluated for a total of 64 responses. Several of the sensors responded positively to the fecal odor, with the formaldehyde, hydrogen sulfide, and ammonia sensors featuring the highest signal to noise ratio. A positive trend was observed between the sensors' responses and the concentration of the odorant and with odor intensity, but no clear correspondence with dilution to threshold (D/T) values was found. Selected sensors were responsive both above and below the intensity values used as the cutoff for offensive odor, suggesting the possibility of using those sensors to differentiate odor offensiveness based just on the magnitude of their response. The specificity of the sensors suggested that discrimination between the selected non-fecal and fecal odors was possible. This study demonstrates that some of the evaluated sensors could be used to assemble a low-cost malodor warning system.

6.
Sci Total Environ ; 713: 136706, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32019042

RESUMO

4.2 billion people live without access to safely managed sanitation services. This report describes the field testing of an onsite prototype system designed to treat blackwater from a single flush toilet and reuse of the treated effluent for flushing. The system passes wastewater through a solid-liquid separator followed by settling tanks and granular activated carbon columns into an electrochemical reactor that oxidizes chloride salts from urine to generate chlorine to remove pathogens. The objectives of the study were to verify the functionality of the system (previously demonstrated in the laboratory) under realistic use conditions, to identify maintenance requirements, and to make a preliminary assessment of the system's user acceptability. The prototype was installed in a women's workplace and residential toilet block in Coimbatore, India, and tested over a period of 10 months. The treated water met stringent disinfection threshold for both E. coli and helminth eggs and produced a clear, colorless effluent that met or nearly met local and international discharge standards for non-sewered sanitation systems. The effluent had an average chemical oxygen demand of 81 mg/L, total suspended solids of 11 mg/L, and reduction of total nitrogen by 65%. These tests determined the recommended service lifetimes and maintenance intervals for key system components including the electrochemical cell, granular activated carbon columns, and solid-liquid separator. User feedback regarding the use of treated blackwater as flush water was positive. These findings will inform the design and implementation of next-generation systems currently under development.

7.
Sci Total Environ ; 703: 135469, 2020 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-31732183

RESUMO

Innovations that enable cost-effective and resource-conserving treatment of human waste are required for the 4.2 billion people in the world who currently lack safe and reliable sanitation services. Onsite treatment and reuse of blackwater is one strategy towards this end, greatly reducing the need to transport wastewater over long distances either via sewers or trucks. Here, we report on the field testing of a prototype onsite blackwater treatment system conducted over a period of 8 months. The system was connected to a women's toilet in a public communal ablution block located in an informal settlement near Durban, South Africa. Liquid waste was treated by separation and diversion of large solids, settling of suspended solids, and filtration through activated carbon prior to disinfection by electrochemical oxidation. System performance was monitored daily by measurement of chemical and physical water quality parameters onsite and confirmed by periodic detailed analysis of chemical and biological parameters at an offsite lab. Daily monitoring of system performance indicated that the effluent had minimal color and turbidity (maximum 90 Pt/Co units and 6.48 NTU, respectively), and consistent evolution of chlorine as blackwater passed through the system. Weekly offsite analysis confirmed that the system consistently inactivated pathogens (E. coli and coliforms) and reduced chemical oxygen demand and total suspended solids to meet ISO 30500 category B standards. Significant reductions in total nitrogen load were also observed, though these reductions often fell short of the 70% reduction required by ISO 30500. No significant reduction in total phosphorus was observed. Maintenance requirements were identified, and the resilience of the system to restart following a prolonged shutdown was demonstrated, but significant improvements are required in the design of the solid/liquid separation mechanism for application of this system in a wiping culture.


Assuntos
Características da Família , Eliminação de Resíduos Líquidos/métodos , África do Sul , Águas Residuárias
8.
Biol Psychol ; 142: 90-99, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30648545

RESUMO

One of the core diagnostic features of autism spectrum disorder (ASD) is engagement in stereotypical motor movements, although the etiology of this repetitive behavior is unknown. Since the 1960s, it has been hypothesized that stereotypical motor movements serve a homeostatic regulation function, and thereby a putative coupling mechanism to cardiovascular arousal. However, to date, surprisingly few reports explicitly assess cardio-somatic coupling and stereotypical motor movements. The present exploratory study investigates coupling of stereotypical body rocking and hand flapping to heart rate and heart rate variability (HRV) in a convenience sample (n = 10) of children and young adults with moderate to profound ASD. Motor movements were recorded via video and three-axis accelerometry, and simultaneous electrocardiographic signals were obtained to determine cardiovascular indices at or around the onset of naturalistically occurring stereotypy. Analysis of the heart rate revealed both repetitive body rocking and hand flapping in particular were found to associate with a strikingly similar cardiovascular pattern of acceleration and deceleration unrelated to physical demands associated with the movements themselves. Furthermore, neither type of stereotypical movement provoked directional change in heart rate variability. The implications of these results and opportunities for future research are discussed.


Assuntos
Transtorno do Espectro Autista/fisiopatologia , Frequência Cardíaca/fisiologia , Atividade Motora/fisiologia , Comportamento Estereotipado/fisiologia , Transtorno de Movimento Estereotipado/fisiopatologia , Acelerometria , Adolescente , Transtorno do Espectro Autista/psicologia , Eletrocardiografia , Feminino , Mãos/fisiopatologia , Humanos , Masculino , Transtorno de Movimento Estereotipado/psicologia , Adulto Jovem
9.
J Water Health ; 16(1): 34-43, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29424716

RESUMO

Water quality and sanitation are inextricably linked to prevalence and control of soil-transmitted helminth infections, a public health concern in resource-limited settings. India bears a large burden of disease associated with poor sanitation. Transformative onsite sanitation technologies are being developed that feature elimination of pathogens including helminth eggs in wastewater treatment. We are conducting third-party testing of multiple sanitation technology systems in Coimbatore (Tamil Nadu) India. To ensure stringent testing of the pathogen removal ability of sanitation technologies, the presence of helminth eggs in wastewater across the town of Coimbatore was assessed. Wastewater samples from existing test sites as well as desludging trucks servicing residential and non-residential septic tanks, were collected. The AmBic methodology (based on washing, sieving, sedimenting and floating) was used for helminth egg isolation. We tested 29 different source samples and found a 52% prevalence of potentially infective helminth eggs. Identification and enumeration of helminth species is reported against the septage source (private residential vs. shared toilet facility) and total solids content. Trichuris egg counts were higher than those of hookworm and Ascaris from desludging trucks, whereas hookworm egg counts were higher in fresh wastewater samples. Surprisingly, no correlation between soil transmitted helminth eggs and total solids was observed.


Assuntos
Fezes/parasitologia , Helmintíase/epidemiologia , Helmintíase/transmissão , Contagem de Ovos de Parasitas/métodos , Solo/parasitologia , Águas Residuárias/parasitologia , Animais , Humanos , Índia/epidemiologia , Saneamento , Toaletes
10.
Gates Open Res ; 2: 52, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-32803126

RESUMO

Background: Transformative sanitation technologies aim to treat fecal sludge (FS) by thermal processes and recover resources from it. There is a paucity of data describing the relevant properties of FS as viable feedstock for thermal treatment in major geographical target areas, such as India. Methods: This study characterized FS collected from septic tanks in two cities located in the Indian southern state of Tamil Nadu. FS samples were obtained at the point of discharge from trucks in Tiruppur (n=85 samples) and Coimbatore (n=50 samples). Additionally, biosolids obtained from sewage treatment plants (STP) in the cities of Coimbatore and Madurai were characterized. Proximate and ultimate analysis as conducted by the fuel industry was carried out. Results: The average higher heating value (HHV) across all FS samples in Tiruppur (13.4 MJ/kg) was much higher than the value for FS in Coimbatore (5.4 MJ/kg), which was partially attributed to the high ash content of 69% in the latter samples.  The HHV in the biosolids samples ranged between 10 and 12.2 MJ/Kg. The average total solids (TS) content for FS was 3.3% and 2.0% for Tiruppur and Coimbatore respectively, while the median TS content for the two cities was 2.3% and 1.2%. The heavy metal content of the ash was found to be below the thresholds for land disposal. Conclusions: This is one of the first studies that has systematically characterized fecal sludge in cities in India and determined its calorific content. We expect these data to serve as input data in the design of thermal processes for fecal sludge treatment.

11.
J Am Soc Mass Spectrom ; 29(2): 360-372, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29052038

RESUMO

Despite many potential applications, miniature mass spectrometers have had limited adoption in the field due to the tradeoff between throughput and resolution that limits their performance relative to laboratory instruments. Recently, a solution to this tradeoff has been demonstrated by using spatially coded apertures in magnetic sector mass spectrometers, enabling throughput and signal-to-background improvements of greater than an order of magnitude with no loss of resolution. This paper describes a proof of concept demonstration of a cycloidal coded aperture miniature mass spectrometer (C-CAMMS) demonstrating use of spatially coded apertures in a cycloidal sector mass analyzer for the first time. C-CAMMS also incorporates a miniature carbon nanotube (CNT) field emission electron ionization source and a capacitive transimpedance amplifier (CTIA) ion array detector. Results confirm the cycloidal mass analyzer's compatibility with aperture coding. A >10× increase in throughput was achieved without loss of resolution compared with a single slit instrument. Several areas where additional improvement can be realized are identified. Graphical Abstract ᅟ.

12.
J Autism Dev Disord ; 48(5): 1458-1466, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29164446

RESUMO

Repetitive sensory motor behaviors are a direct target for clinical treatment and a potential treatment endpoint for individuals with intellectual or developmental disabilities. By removing the burden associated with video annotation or direct observation, automated detection of stereotypy would allow for longer term monitoring in ecologic settings. We report automated detection of common stereotypical motor movements using commercially available accelerometers affixed to the body and a generalizable detection algorithm. The method achieved a sensitivity of 80% for body rocking and 93% for hand flapping without individualized algorithm training or foreknowledge of subject's specific movements. This approach is well-suited for implementation in a continuous monitoring system outside of a clinical setting.


Assuntos
Acelerometria/métodos , Deficiências do Desenvolvimento/fisiopatologia , Deficiência Intelectual/fisiopatologia , Atividade Motora/fisiologia , Transtorno de Movimento Estereotipado/fisiopatologia , Adolescente , Algoritmos , Criança , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/psicologia , Feminino , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/psicologia , Masculino , Movimento/fisiologia , Comportamento Estereotipado/fisiologia , Transtorno de Movimento Estereotipado/psicologia , Adulto Jovem
13.
Altern Lab Anim ; 45(3): 117-158, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28816053

RESUMO

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.


Assuntos
Fumar/efeitos adversos , Produtos do Tabaco/efeitos adversos , Testes de Toxicidade/métodos , Aerossóis , Animais , Sistemas Eletrônicos de Liberação de Nicotina/efeitos adversos , Humanos , Técnicas In Vitro , Especificidade da Espécie , Estados Unidos , United States Food and Drug Administration
14.
ALTEX ; 34(2): 301-310, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27846345

RESUMO

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, "organotypic" cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.


Assuntos
Técnicas de Cultura de Células , Simulação por Computador , Biologia de Sistemas , Alternativas aos Testes com Animais , Animais , Técnicas de Cultura de Células/métodos , Substâncias Perigosas/toxicidade , Humanos , Dispositivos Lab-On-A-Chip , Medição de Risco
15.
Biomicrofluidics ; 9(6): 061102, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26594261

RESUMO

We report a microfluidic blood-brain barrier model that enables both physiological shear stress and optical transparency throughout the device. Brain endothelial cells grown in an optically transparent membrane-integrated microfluidic device were able to withstand physiological fluid shear stress using a hydrophilized polytetrafluoroethylene nanoporous membrane instead of the more commonly used polyester membrane. A functional three-dimensional microfluidic co-culture model of the neurovascular unit is presented that incorporates astrocytes in a 3D hydrogel and enables physiological shear stress on the membrane-supported endothelial cell layer.

16.
Toxicol Appl Pharmacol ; 288(2): 249-57, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26232523

RESUMO

Microelectrode arrays (MEAs) recording extracellular field potentials of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) provide a rich data set for functional assessment of drug response. The aim of this work is the development of a method for a systematic analysis of arrhythmia using MEAs, with emphasis on the development of six parameters accounting for different types of cardiomyocyte signal irregularities. We describe a software approach to carry out such analysis automatically including generation of a heat map that enables quick visualization of arrhythmic liability of compounds. We also implemented signal processing techniques for reliable extraction of the repolarization peak for field potential duration (FPD) measurement even from recordings with low signal to noise ratios. We measured hiPS-CM's on a 48 well MEA system with 5minute recordings at multiple time points (0.5, 1, 2 and 4h) after drug exposure. We evaluated concentration responses for seven compounds with a combination of hERG, QT and clinical proarrhythmia properties: Verapamil, Ranolazine, Flecainide, Amiodarone, Ouabain, Cisapride, and Terfenadine. The predictive utility of MEA parameters as surrogates of these clinical effects were examined. The beat rate and FPD results exhibited good correlations with previous MEA studies in stem cell derived cardiomyocytes and clinical data. The six-parameter arrhythmia assessment exhibited excellent predictive agreement with the known arrhythmogenic potential of the tested compounds, and holds promise as a new method to predict arrhythmic liability.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Ensaios de Triagem em Larga Escala/instrumentação , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Microeletrodos , Miócitos Cardíacos/efeitos dos fármacos , Testes de Toxicidade/instrumentação , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Automação Laboratorial , Diferenciação Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Desenho de Equipamento , Frequência Cardíaca/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Medição de Risco , Processamento de Sinais Assistido por Computador , Razão Sinal-Ruído , Software , Fatores de Tempo , Testes de Toxicidade/métodos
17.
Brain Res ; 1608: 167-76, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25721792

RESUMO

Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)ß1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFß1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.


Assuntos
Astrócitos/efeitos dos fármacos , Encéfalo/citologia , Células Endoteliais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Animais , Astrócitos/fisiologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Transformada , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Imageamento Tridimensional , Camundongos , Fatores de Tempo
18.
Lab Chip ; 14(17): 3349-58, 2014 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-25000964

RESUMO

Microfluidic cell cultures enable investigation of complex physiological tissue properties and functionalities. For convenience, they are often implemented with immortalized cell lines, but primary cells more closely approximate the in vivo biology. Our aim was to develop a biomimetic microfluidic model of the human airway using all primary cells. The model is comprised of airway epithelial cells cultured at an air-liquid interface, lung fibroblasts and polarized microvascular endothelial cells, respectively positioned in three vertically stacked, individually accessible compartments separated by nanoporous membranes. We report device fabrication, a gravity fed microfluidic system, and culture medium able to support functional co-cultures of all three primary human cell types. As characterized by imaging and permeability measurements, airway epithelial cells in microfluidic devices displayed mucociliary differentiation and barrier function. Subjacent fibroblasts and microvascular endothelial cells were added under conditions enabling co-culture for at least 5 days. Microfluidic airway models based on primary human cells in a relevant biomimetic configuration will improve physiological relevance and will enable novel disease modeling and drug development studies.


Assuntos
Biomimética , Traqueia/citologia , Técnicas de Cocultura , Meios de Cultura , Humanos , Microfluídica , Modelos Biológicos
19.
Proc Natl Acad Sci U S A ; 110(40): 16157-62, 2013 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24043791

RESUMO

Severe acute respiratory syndrome with high mortality rates (~50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ~0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3-5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3-5 showed 1-1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.


Assuntos
Doenças Transmissíveis Emergentes/virologia , Coronavirus/genética , DNA Complementar/genética , Síndrome Respiratória Aguda Grave/virologia , Northern Blotting , Western Blotting , Células Cultivadas , Primers do DNA/genética , Dipeptidil Peptidase 4/metabolismo , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Proteínas Luminescentes , Oriente Médio , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase em Tempo Real , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/fisiologia , Ligação Viral , Replicação Viral/fisiologia
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