RESUMO
The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved â¼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , Telefone Celular/instrumentação , Imagem Óptica/métodos , RNA Viral/análise , Carga Viral/métodos , Animais , Teste de Ácido Nucleico para COVID-19/economia , Teste de Ácido Nucleico para COVID-19/instrumentação , Sistemas CRISPR-Cas , Linhagem Celular , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Nasofaringe/virologia , Imagem Óptica/instrumentação , Fosfoproteínas/genética , Testes Imediatos , Interferência de RNA , RNA Viral/genética , Sensibilidade e Especificidade , Carga Viral/economia , Carga Viral/instrumentaçãoRESUMO
BACKGROUND: Implementation of an ivermectin-based community treatment strategy for the elimination of onchocerciasis or lymphatic filariasis has been delayed in Central Africa because of the occurrence of serious adverse events, including death, in persons with high levels of circulating Loa loa microfilariae. The LoaScope, a field-friendly diagnostic tool to quantify L. loa microfilariae in peripheral blood, enables rapid, point-of-care identification of persons at risk for serious adverse events. METHODS: A test-and-not-treat strategy was used in the approach to ivermectin treatment in the Okola health district in Cameroon, where the distribution of ivermectin was halted in 1999 after the occurrence of fatal events related to L. loa infection. The LoaScope was used to identify persons with an L. loa microfilarial density greater than 20,000 microfilariae per milliliter of blood, who were considered to be at risk for serious adverse events, and exclude them from ivermectin distribution. Active surveillance for posttreatment adverse events was performed daily for 6 days. RESULTS: From August through October 2015, a total of 16,259 of 22,842 persons 5 years of age or older (71.2% of the target population) were tested for L. loa microfilaremia. Among the participants who underwent testing, a total of 15,522 (95.5%) received ivermectin, 340 (2.1%) were excluded from ivermectin distribution because of an L. loa microfilarial density above the risk threshold, and 397 (2.4%) were excluded because of pregnancy or illness. No serious adverse events were observed. Nonserious adverse events were recorded in 934 participants, most of whom (67.5%) had no detectable L. loa microfilariae. CONCLUSIONS: The LoaScope-based test-and-not-treat strategy enabled the reimplementation of community-wide ivermectin distribution in a heretofore "off limits" health district in Cameroon and is a potentially practical approach to larger-scale ivermectin treatment for lymphatic filariasis and onchocerciasis in areas where L. loa infection is endemic. (Funded by the Bill and Melinda Gates Foundation and others.).
Assuntos
Antiparasitários/uso terapêutico , Doenças Endêmicas , Ivermectina/uso terapêutico , Loa/isolamento & purificação , Loíase/diagnóstico , Oncocercose/tratamento farmacológico , Adolescente , Adulto , Idoso , Animais , Antiparasitários/efeitos adversos , Sangue/parasitologia , Camarões , Criança , Filariose Linfática/complicações , Filariose Linfática/tratamento farmacológico , Feminino , Humanos , Ivermectina/efeitos adversos , Modelos Logísticos , Loíase/complicações , Loíase/epidemiologia , Masculino , Microfilárias/isolamento & purificação , Microscopia de Vídeo/instrumentação , Pessoa de Meia-Idade , Oncocercose/complicaçõesRESUMO
BACKGROUND: The neurologic emergency department (neuro ED) at our medical center is staffed by emergency medicine physicians who have specialized neuroscience training and give intravenous (IV) tissue plasminogen activator (tPA) independently for acute ischemic stroke patients. Door-to-needle (DTN) times, discharge location, and discharge National Institute of Health Stroke Scale (NIHSS) scores were studied between the neuro ED and main emergency department (ED) with the hypothesis that all measures would be better in the neuro ED group. METHODS: This is a retrospective study evaluating DTN time, discharge outcomes, and discharge location in acute stroke patients who received IV tPA at our comprehensive stroke center. These outcome measures were compared between patients who were evaluated and treated in our neuro ED to those treated in our main ED. RESULTS: From 2012 to 2014, 67 acute stroke patients received IV tPA in our ED. Thirty-five patients were evaluated in the neuro ED, and 32, in the main ED. Average DTN times were significantly faster in the neuro ED at 35 minutes, compared to main ED DTN times of 83 minutes. Discharge NIHSS score was significantly lower, and more patients were discharged to home in the neuro ED group compared to the main ED group. CONCLUSIONS: Trained neuro ED physicians can safely give IV tPA independently for stroke patients with improved DTN times, lower discharge NIHSS, and higher likelihood of being discharged to home compared to the main ED physicians who used teleneurology consultation. This suggests utility in training emergency medicine physicians to administer tPA independently based on clinical practice guidelines.
Assuntos
Serviço Hospitalar de Emergência/estatística & dados numéricos , Neurociências/educação , Melhoria de Qualidade , Acidente Vascular Cerebral/tratamento farmacológico , Ativador de Plasminogênio Tecidual/uso terapêutico , Idoso , Medicina de Emergência/educação , Medicina de Emergência/normas , Medicina de Emergência/estatística & dados numéricos , Serviço Hospitalar de Emergência/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Melhoria de Qualidade/organização & administração , Melhoria de Qualidade/estatística & dados numéricos , Estudos Retrospectivos , Acidente Vascular Cerebral/diagnóstico , Fatores de Tempo , Resultado do Tratamento , Recursos HumanosRESUMO
Catatonia was first described by a German psychiatrist, Karl Kahlbaum, in 1874. It is a behavioral syndrome marked by an inability to move normally, which can occur in the context of many underlying general medical and psychiatric disorders. A wide variety of neurologic, metabolic, drug-induced, and psychiatric causes of catatonia have been reported. We present a unique case of late onset catatonia in a 56-year-old man with no prior medical or psychiatric history initially presenting with stroke-like symptoms. The patient was awake and alert, with spontaneous eye opening, but completely nonverbal and not following any commands. Specifically, the patient demonstrated stupor, catalepsy, mutism, and negativism. After extensive emergency department testing, including negative computed tomography head, negative magnetic resonance imaging brain, negative electroencephalogram, and normal laboratory results, the patient was diagnosed with new-onset bipolar disorder with depressive features presenting as catatonia. Recognizing catatonia is important because it may be caused or exacerbated by treatment of the underlying disorder. Failure to institute treatment early in the course of catatonia is associated with a poor prognosis.
Assuntos
Transtorno Bipolar/diagnóstico , Catatonia/diagnóstico , Transtorno Bipolar/complicações , Catalepsia/etiologia , Catatonia/etiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutismo/etiologia , Estupor/etiologiaRESUMO
PURPOSE: To evaluate the efficacy of a novel mobile phone application that calculates superior visual field defects on Goldmann visual field charts. METHODS: Experimental study in which the mobile phone application and 14 oculoplastic surgeons interpreted the superior visual field defect in 10 Goldmann charts. Percent error of the mobile phone application and the oculoplastic surgeons' estimates were calculated compared with computer software computation of the actual defects. Precision and time efficiency of the application were evaluated by processing the same Goldmann visual field chart 10 repeated times. RESULTS: The mobile phone application was associated with a mean percent error of 1.98% (95% confidence interval[CI], 0.87%-3.10%) in superior visual field defect calculation. The average mean percent error of the oculoplastic surgeons' visual estimates was 19.75% (95% CI, 14.39%-25.11%). Oculoplastic surgeons, on average, underestimated the defect in all 10 Goldmann charts. There was high interobserver variance among oculoplastic surgeons. The percent error of the 10 repeated measurements on a single chart was 0.93% (95% CI, 0.40%-1.46%). The average time to process 1 chart was 12.9 seconds (95% CI, 10.9-15.0 seconds). CONCLUSIONS: The mobile phone application was highly accurate, precise, and time-efficient in calculating the percent superior visual field defect using Goldmann charts. Oculoplastic surgeon visual interpretations were highly inaccurate, highly variable, and usually underestimated the field vision loss.
Assuntos
Blefaroptose/diagnóstico , Telefone Celular , Aplicativos Móveis , Transtornos da Visão/diagnóstico , Testes de Campo Visual/instrumentação , Campos Visuais , Blefaroplastia , Blefaroptose/cirurgia , Humanos , Variações Dependentes do Observador , Reprodutibilidade dos Testes , Inquéritos e Questionários , Testes de Campo Visual/métodosRESUMO
Schistosomiasis infections continue to impact African settings disproportionately, and there is an urgent need for novel tools to evaluate infection control and elimination strategies at the community level. Mobile phone microscopes are portable and semiautomated devices with multiple applications for screening neglected tropical diseases. In a community-based schistosomiasis screening program in Azaguié, Côte d'Ivoire, mobile phone microscopy demonstrated a sensitivity of 85.7% (95% CI: 69.7-95.2%) and specificity of 93.3% (95% CI: 87.7-96.9%) for Schistosoma haematobium identification compared with conventional light microscopy, and 95% sensitivity (95% CI: 74.1-99.8%) with egg concentrations of five or more per 10 mL of urine. Mobile phone microscopy is a promising tool for schistosomiasis control and elimination efforts.
Assuntos
Telefone Celular , Esquistossomose , Humanos , Animais , Schistosoma haematobium , Microscopia , Côte d'Ivoire/epidemiologia , Esquistossomose/diagnósticoRESUMO
Schistosoma haematobium continues to pose a significant public health burden despite ongoing global control efforts. One of several barriers to sustained control (and ultimately elimination) is the lack of access to highly sensitive diagnostic or screening tools that are inexpensive, rapid, and can be used at the point of sample collection. Here, we report an automated point-of-care diagnostic based on mobile phone microscopy that rapidly images and identifies S. haematobium eggs in urine samples. Parasite eggs are filtered from urine within a specialized, inexpensive cartridge that is then automatically imaged by the mobile phone microscope (the "SchistoScope"). Parasite eggs are captured at a constriction point in the tapered cartridge for easy imaging, and the automated quantification of eggs is obtained upon analysis of the images by an algorithm. We demonstrate S. haematobium egg detection with greater than 90% sensitivity and specificity using this device compared with the field gold standard of conventional filtration and microscopy. With simple sample preparation and image analysis on a mobile phone, the SchistoScope combines the diagnostic performance of conventional microscopy with the analytic performance of an expert technician. This portable device has the potential to provide rapid and quantitative diagnosis of S. haematobium to advance ongoing control efforts.
RESUMO
Early detection of oral cancer necessitates a minimally invasive, tissue-specific diagnostic tool that facilitates screening/surveillance. Brush biopsy, though minimally invasive, demands skilled cyto-pathologist expertise. In this study, we explored the clinical utility/efficacy of a tele-cytology system in combination with Artificial Neural Network (ANN) based risk-stratification model for early detection of oral potentially malignant (OPML)/malignant lesion. A portable, automated tablet-based tele-cytology platform capable of digitization of cytology slides was evaluated for its efficacy in the detection of OPML/malignant lesions (n = 82) in comparison with conventional cytology and histology. Then, an image pre-processing algorithm was established to segregate cells, ANN was trained with images (n = 11,981) and a risk-stratification model developed. The specificity, sensitivity and accuracy of platform/ stratification model were computed, and agreement was examined using Kappa statistics. The tele-cytology platform, Cellscope, showed an overall accuracy of 84-86% with no difference between tele-cytology and conventional cytology in detection of oral lesions (kappa, 0.67-0.72). However, OPML could be detected with low sensitivity (18%) in accordance with the limitations of conventional cytology. The integration of image processing and development of an ANN-based risk stratification model improved the detection sensitivity of malignant lesions (93%) and high grade OPML (73%), thereby increasing the overall accuracy by 30%. Tele-cytology integrated with the risk stratification model, a novel strategy established in this study, can be an invaluable Point-of-Care (PoC) tool for early detection/screening in oral cancer. This study hence establishes the applicability of tele-cytology for accurate, remote diagnosis and use of automated ANN-based analysis in improving its efficacy.
Assuntos
Citodiagnóstico/métodos , Detecção Precoce de Câncer , Neoplasias Bucais/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Telemedicina/métodos , Algoritmos , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Redes Neurais de Computação , Medição de Risco , Sensibilidade e EspecificidadeRESUMO
Ivermectin treatment can cause central nervous system adverse events (CNS-AEs) in persons with very high-density Loa loa microfilaremia (≥ 30,000 mf/mL blood). Hypoendemic onchocerciasis areas where L. loa is endemic have been excluded from ivermectin mass drug administration programs (MDA) because of the concern for CNS AEs. The rapid assessment procedure for L. loa (RAPLOA) is a questionnaire survey to assess history of eye worm. If ≥ 40% of respondents report eye worm, this correlates with ≥ 2% prevalence of very high-density loiasis microfilaremia, posing an unacceptable risk of CNS-AEs after MDA. In 2016, we conducted a L. loa study in 110 ivermectin-naïve, suspected onchocerciasis hypoendemic villages in southern Nigeria. In previous RAPLOA surveys these villages had prevalences between 10% and 67%. We examined 10,605 residents using the LoaScope, a cell phone-based imaging device for rapidly determining the microfilaria (mf) density of L. loa infections. The mean L. loa village mf prevalence was 6.3% (range 0-29%) and the mean individual mf count among positives was 326 mf/mL. The maximum individual mf count was only 11,429 mf/mL, and among 2,748 persons sampled from the 28 villages with ≥ 40% RAPLOA, the ≥ 2% threshold of very high Loa mf density could be excluded with high statistical confidence (P < 0.01). These findings indicate that ivermectin MDA can be delivered in this area with extremely low risk of L. loa-related CNS-AEs. We also concluded that in Nigeria the RAPLOA survey methodology is not predictive of ≥ 2% prevalence of very high-density L. loa microfilaremia.
Assuntos
Doenças Endêmicas/estatística & dados numéricos , Loa/isolamento & purificação , Loíase/epidemiologia , Carga Parasitária , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Olho , Feminino , Filaricidas/uso terapêutico , Humanos , Ivermectina/uso terapêutico , Loa/patogenicidade , Loíase/diagnóstico , Loíase/parasitologia , Masculino , Administração Massiva de Medicamentos/métodos , Nigéria/epidemiologia , Prevalência , População Rural , Inquéritos e QuestionáriosRESUMO
Oral cancer is the most common type of cancer among men in India and other countries in South Asia. Late diagnosis contributes significantly to this mortality, highlighting the need for effective and specific point-of-care diagnostic tools. The same regions with high prevalence of oral cancer have seen extensive growth in mobile phone infrastructure, which enables widespread access to telemedicine services. In this work, we describe the evaluation of an automated tablet-based mobile microscope as an adjunct for telemedicine-based oral cancer screening in India. Brush biopsy, a minimally invasive sampling technique was combined with a simplified staining protocol and a tablet-based mobile microscope to facilitate local collection of digital images and remote evaluation of the images by clinicians. The tablet-based mobile microscope (CellScope device) combines an iPad Mini with collection optics, LED illumination and Bluetooth-controlled motors to scan a slide specimen and capture high-resolution images of stained brush biopsy samples. Researchers at the Mazumdar Shaw Medical Foundation (MSMF) in Bangalore, India used the instrument to collect and send randomly selected images of each slide for telepathology review. Evaluation of the concordance between gold standard histology, conventional microscopy cytology, and remote pathologist review of the images was performed as part of a pilot study of mobile microscopy as a screening tool for oral cancer. Results indicated that the instrument successfully collected images of sufficient quality to enable remote diagnoses that show concordance with existing techniques. Further studies will evaluate the effectiveness of oral cancer screening with mobile microscopy by minimally trained technicians in low-resource settings.
Assuntos
Telefone Celular , Detecção Precoce de Câncer/métodos , Microscopia/métodos , Neoplasias Bucais/diagnóstico , Adulto , Idoso , Automação , Demografia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Índia , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/patologia , Projetos Piloto , Sensibilidade e Especificidade , Interface Usuário-Computador , Adulto JovemRESUMO
BACKGROUND: Handheld light microscopy using compact optics and mobile phones may improve the quality of health care in resource-constrained settings by enabling access to prompt and accurate diagnosis. METHODOLOGY: Laboratory technicians were trained to operate two handheld diagnostic devices (Newton Nm1 microscope and a clip-on version of the mobile phone-based CellScope). The accuracy of these devices was compared to conventional light microscopy for the diagnosis of Schistosoma haematobium, S. mansoni, and intestinal protozoa infection in a community-based survey in rural Côte d'Ivoire. One slide of 10 ml filtered urine and a single Kato-Katz thick smear from 226 individuals were subjected to the Newton Nm1 microscope and CellScope for detection of Schistosoma eggs and compared to conventional microscopy. Additionally, 121 sodium acetate-acetic acid-formalin (SAF)-fixed stool samples were examined by the Newton Nm1 microscope and compared to conventional microscopy for the diagnosis of intestinal protozoa. PRINCIPAL FINDINGS: The prevalence of S. haematobium, S. mansoni, Giardia intestinalis, and Entamoeba histolytica/E. dispar, as determined by conventional microscopy, was 39.8%, 5.3%, 20.7%, and 4.9%, respectively. The Newton Nm1 microscope had diagnostic sensitivities for S. mansoni and S. haematobium infection of 91.7% (95% confidence interval (CI) 59.8-99.6%) and 81.1% (95% CI 71.2-88.3%), respectively, and specificities of 99.5% (95% CI 97.0-100%) and 97.1% (95% CI 92.2-99.1%), respectively. The CellScope demonstrated sensitivities for S. mansoni and S. haematobium of 50.0% (95% CI 25.4-74.6%) and 35.6% (95% CI 25.9-46.4%), respectively, and specificities of 99.5% (95% CI 97.0-100%) and 100% (95% CI 86.7-100%), respectively. For G. intestinalis and E. histolytica/E. dispar, the Newton Nm1 microscope had sensitivity of 84.0% (95% CI 63.1-94.7%) and 83.3% (95% CI 36.5-99.1%), respectively, and 100% specificity. CONCLUSIONS/SIGNIFICANCE: Handheld diagnostic devices can be employed in community-based surveys in resource-constrained settings after minimal training of laboratory technicians to diagnose intestinal parasites.
Assuntos
Telefone Celular , Enteropatias Parasitárias/diagnóstico , Microscopia/instrumentação , Microscopia/métodos , Infecções por Protozoários/diagnóstico , Esquistossomose/diagnóstico , Animais , Côte d'Ivoire/epidemiologia , Humanos , Enteropatias Parasitárias/parasitologia , Infecções por Protozoários/epidemiologia , Infecções por Protozoários/parasitologia , Schistosoma haematobium/isolamento & purificação , Schistosoma mansoni/isolamento & purificação , Esquistossomose/epidemiologia , Esquistossomose/parasitologia , Sensibilidade e Especificidade , Solo/parasitologiaRESUMO
We evaluated two novel, portable microscopes and locally acquired, single-ply, paper towels as filter paper for the diagnosis of Schistosoma haematobium infection. The mobile phone-mounted Foldscope and reversed-lens CellScope had sensitivities of 55.9% and 67.6%, and specificities of 93.3% and 100.0%, respectively, compared with conventional light microscopy for diagnosing S. haematobium infection. With conventional light microscopy, urine filtration using single-ply paper towels as filter paper showed a sensitivity of 67.6% and specificity of 80.0% compared with centrifugation for the diagnosis of S. haematobium infection. With future improvements to diagnostic sensitivity, newer generation handheld and mobile phone microscopes may be valuable tools for global health applications.
Assuntos
Telefone Celular , Microscopia/métodos , Schistosoma haematobium , Esquistossomose Urinária/diagnóstico , Adolescente , Animais , Centrifugação , Criança , Gana , Humanos , Microscopia/instrumentação , Contagem de Ovos de Parasitas , Esquistossomose Urinária/urina , Sensibilidade e EspecificidadeRESUMO
We demonstrate the design and application of an add-on device for improving the diagnostic and research capabilities of CellScope--a low-cost, smartphone-based point-of-care microscope. We replace the single LED illumination of the original CellScope with a programmable domed LED array. By leveraging recent advances in computational illumination, this new device enables simultaneous multi-contrast imaging with brightfield, darkfield, and phase imaging modes. Further, we scan through illumination angles to capture lightfield datasets, which can be used to recover 3D intensity and phase images without any hardware changes. This digital refocusing procedure can be used for either 3D imaging or software-only focus correction, reducing the need for precise mechanical focusing during field experiments. All acquisition and processing is performed on the mobile phone and controlled through a smartphone application, making the computational microscope compact and portable. Using multiple samples and different objective magnifications, we demonstrate that the performance of our device is comparable to that of a commercial microscope. This unique device platform extends the field imaging capabilities of CellScope, opening up new clinical and research possibilities.
Assuntos
Processamento de Imagem Assistida por Computador/estatística & dados numéricos , Imageamento Tridimensional/instrumentação , Microscopia/instrumentação , Software , Desenho de Equipamento , Análise de Fourier , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Microscopia/métodos , Sistemas Automatizados de Assistência Junto ao Leito , Smartphone/instrumentaçãoRESUMO
Parasitic helminths cause debilitating diseases that affect millions of people in primarily low-resource settings. Efforts to eliminate onchocerciasis and lymphatic filariasis in Central Africa through mass drug administration have been suspended because of ivermectin-associated serious adverse events, including death, in patients infected with the filarial parasite Loa loa. To safely administer ivermectin for onchocerciasis or lymphatic filariasis in regions co-endemic with L. loa, a strategy termed "test and (not) treat" has been proposed whereby those with high levels of L. loa microfilariae (>30,000/ml) that put them at risk for life-threatening serious adverse events are identified and excluded from mass drug administration. To enable this, we developed a mobile phone-based video microscope that automatically quantifies L. loa microfilariae in whole blood loaded directly into a small glass capillary from a fingerprick without the need for conventional sample preparation or staining. This point-of-care device automatically captures and analyzes videos of microfilarial motion in whole blood using motorized sample scanning and onboard motion detection, minimizing input from health care workers and providing a quantification of microfilariae per milliliter of whole blood in under 2 min. To validate performance and usability of the mobile phone microscope, we tested 33 potentially Loa-infected patients in Cameroon and confirmed that automated counts correlated with manual thick smear counts (94% specificity; 100% sensitivity). Use of this technology to exclude patients from ivermectin-based treatment at the point of care in Loa-endemic regions would allow resumption/expansion of mass drug administration programs for onchocerciasis and lymphatic filariasis in Central Africa.
Assuntos
Telefone Celular , Filariose Linfática/diagnóstico , Loa/isolamento & purificação , Microscopia de Vídeo/métodos , Oncocercose/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , África Central , Algoritmos , Animais , Automação , Camarões , Reações Falso-Negativas , Processamento de Imagem Assistida por Computador , Movimento (Física) , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The increasing capabilities and ubiquity of mobile phones and their associated digital cameras offer the possibility of extending low-cost, portable diagnostic microscopy to underserved and low-resource areas. However, mobile phone microscopes created by adding magnifying optics to the phone's camera module have been unable to make use of the full image sensor due to the specialized design of the embedded camera lens, exacerbating the tradeoff between resolution and field of view inherent to optical systems. This tradeoff is acutely felt for diagnostic applications, where the speed and cost of image-based diagnosis is related to the area of the sample that can be viewed at sufficient resolution. Here we present a simple and low-cost approach to mobile phone microscopy that uses a reversed mobile phone camera lens added to an intact mobile phone to enable high quality imaging over a significantly larger field of view than standard microscopy. We demonstrate use of the reversed lens mobile phone microscope to identify red and white blood cells in blood smears and soil-transmitted helminth eggs in stool samples.
Assuntos
Telefone Celular/instrumentação , Microscopia/instrumentação , Animais , Células Sanguíneas/citologia , Telefone Celular/economia , Desenho de Equipamento , Fezes/parasitologia , Helmintos/isolamento & purificação , Humanos , Lentes/economia , Microscopia/economiaRESUMO
Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.
Assuntos
Movimento Celular/fisiologia , Drosophila melanogaster/citologia , Adesões Focais/fisiologia , Mecanotransdução Celular/fisiologia , Animais , Adesão Celular/genética , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/genética , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Mecanotransdução Celular/genética , Microscopia de Fluorescência , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Interferência de RNA , Imagem com Lapso de Tempo , Vitronectina/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismoRESUMO
Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (~14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca(2+)-dependent adhesion in DE-cadherin-expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca(2+)-independent cell-cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein-protein interactions that regulate the levels of the core cadherin-catenin complex and coordinate cadherin-mediated cell-cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin-Darby canine kidney mammalian epithelial cell-cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell-substrate interactions, and nuclear and cytoplasmic signaling.
Assuntos
Caderinas/fisiologia , Adesão Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Animais , Caderinas/metabolismo , Adesão Celular/fisiologia , Células Cultivadas , Cães , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/citologia , Técnicas de Silenciamento de Genes , Genoma de Inseto , Células Madin Darby de Rim Canino , Interferência de RNARESUMO
Recent technological advances in microscopy have enabled cell-based whole genome screens, but the analysis of the vast amount of image data generated by such screens usually proves to be rate limiting. In this study, we performed a whole genome RNA interference (RNAi) screen to uncover genes that affect spreading of Drosophila melanogaster S2 cells using several computational methods for analyzing the image data in an automated manner. Expected genes in the Scar-Arp2/3 actin nucleation pathway were identified as well as casein kinase I, which had a similar morphological RNAi signature. A distinct nonspreading morphological phenotype was identified for genes involved in membrane secretion or synthesis. In this group, we identified a new secretory peptide and investigated the functions of two poorly characterized endoplasmic reticulum proteins that have roles in secretion. Thus, this genome-wide screen succeeded in identifying known and unexpected proteins that are important for cell spreading, and the computational tools developed in this study should prove useful for other types of automated whole genome screens.
Assuntos
Forma Celular/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Genoma de Inseto/genética , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Imagem Molecular/métodos , Interferência de RNA , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Automação , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Linhagem Celular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Asymmetric positioning of the mitotic spindle before cytokinesis can produce different-sized daughter cells that have distinct fates. Here, we found an asymmetric division in the Caenorhabditis elegans Q neuroblast lineage that began with a centered spindle but generated different-sized daughters, the smaller (anterior) of which underwent apoptosis. During this division, more myosin II accumulated anteriorly, suggesting that asymmetric contractile forces might produce different-sized daughters. Indeed, partial inactivation of anterior myosin by chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and differentiation of the anterior daughter. Thus, the balance of myosin activity on the two sides of a dividing cell can govern the size and fate of the daughters.