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1.
J Vis Exp ; (163)2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-33016946

RESUMO

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease. C. elegans adult neurons that express aggregating proteins can extrude large (~4 µm) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called "exophers" and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases. While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons. Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.


Assuntos
Caenorhabditis elegans/citologia , Técnicas Citológicas/métodos , Neurônios/citologia , Organelas/metabolismo , Animais , Agregação Celular , Humanos
2.
Sci Rep ; 10(1): 13179, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764697

RESUMO

The survival and function of brain cells requires uninterrupted ATP synthesis. Different brain structures subserve distinct neurological functions, and therefore have different energy production/consumption requirements. Typically, mitochondrial function is assessed following their isolation from relatively large amounts of starting tissue, making it difficult to ascertain energy production/failure in small anatomical locations. In order to overcome this limitation, we have developed and optimized a method to measure mitochondrial function in brain tissue biopsy punches excised from anatomically defined brain structures, including white matter tracts. We describe the procedures for maintaining tissue viability prior to performing the biopsy punches, as well as provide guidance for optimizing punch size and the drug doses needed to assess various aspects of mitochondrial respiration. We demonstrate that our method can be used to measure mitochondrial respiration in anatomically defined subfields within the rat hippocampus. Using this method, we present experimental results which show that a mild traumatic brain injury (mTBI, often referred to as concussion) causes differential mitochondrial responses within these hippocampal subfields and the corpus callosum, novel findings that would have been difficult to obtain using traditional mitochondrial isolation methods. Our method is easy to implement and will be of interest to researchers working in the field of brain bioenergetics and brain diseases.


Assuntos
Encéfalo/anatomia & histologia , Encéfalo/citologia , Técnicas Citológicas/métodos , Animais , Lesões Encefálicas/patologia , Respiração Celular , Hipocampo/patologia , Masculino , Mitocôndrias/metabolismo , Ratos , Ratos Sprague-Dawley , Substância Branca/patologia
3.
Nat Commun ; 11(1): 4339, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859909

RESUMO

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.


Assuntos
Técnicas Citológicas/métodos , DNA/química , Microscopia de Fluorescência/métodos , Simulação de Acoplamento Molecular/métodos , Linhagem Celular , Humanos , Processamento de Imagem Assistida por Computador/métodos , Oligonucleotídeos , Coloração e Rotulagem/métodos
4.
J Vis Exp ; (159)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32510501

RESUMO

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.


Assuntos
Comunicação Celular , Técnicas Citológicas/métodos , Agregados Proteicos , Agregação Celular , Células HEK293 , Humanos , Ligantes
5.
Nat Protoc ; 15(7): 2230-2246, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561889

RESUMO

Macrophage phagocytosis can be triggered by diverse receptor-ligand interactions to clear pathogens and dead cells from a host. Many ways of assaying phagocytosis exist that utilize a variety of phagocytic targets with different combinations of receptor-ligand interactions, making comparisons difficult. To study how phagocytosis is affected by specific changes to the target surface, we developed an in vitro assay based on reconstituted membrane-coated target particles to which known molecules can be added. The targets are made by coating glass beads with supported lipid bilayers followed by coupling proteins and other ligands of interest. Composition of the lipid bilayer can be varied to bind and orient specific proteins, incorporate signaling and reporter lipids, and control bilayer fluidity. To quantify phagocytosis, the reconstituted target particles are incubated with macrophages in vitro for a defined period of time, imaged with fluorescence microscopy and analyzed with software that measures the amount of target particle fluorescence within each macrophage. A multi-well plate format can be used for multi-parameter studies (e.g., to investigate how phagocytosis is affected by specific receptor-ligand interactions, ligand density, lipid charge, membrane fluidity and other molecular details). As an example, we demonstrate that antibody-dependent phagocytosis is more efficient for targets with fluid membranes than non-fluid membranes. The assay protocol takes approximately 6 h and requires basic molecular biology, mammalian cell culture and fluorescence microscopy skills. This assay can also be used with other phagocytic and non-phagocytic cells to study the individual or collective roles of receptors and ligands in immune effector function.


Assuntos
Técnicas Citológicas/métodos , Macrófagos/citologia , Fagocitose , Animais , Camundongos , Células RAW 264.7
6.
Sci Rep ; 10(1): 7540, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371955

RESUMO

Large dense-core vesicles (LDCVs) contain a variety of neurotransmitters, proteins, and hormones such as biogenic amines and peptides, together with microRNAs (miRNAs). Isolation of LDCVs is essential for functional studies including vesicle fusion, vesicle acidification, monoamine transport, and the miRNAs stored in LDCVs. Although several methods were reported for purifying LDCVs, the final fractions are significantly contaminated by other organelles, compromising biochemical characterization. Here we isolated LDCVs (chromaffin granules) with high yield and purity from bovine adrenal medulla. The fractionation protocol combines differential and continuous sucrose gradient centrifugation, allowing for reducing major contaminants such as mitochondria. Purified LDCVs show robust acidification by the endogenous V-ATPase and undergo SNARE-mediated fusion with artificial membranes. Interestingly, LDCVs contain specific miRNAs such as miR-375 and miR-375 is stabilized by protein complex against RNase A. This protocol can be useful in research on the biological functions of LDCVs.


Assuntos
Medula Suprarrenal/fisiologia , Técnicas Citológicas/métodos , Animais , Bovinos , Fracionamento Celular , Grânulos Cromafim/metabolismo , Fusão de Membrana , MicroRNAs/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo
9.
Cancer Cytopathol ; 128(5): 317-320, 2020 May.
Artigo em Inglês | MEDLINE | ID: covidwho-38690

RESUMO

The 2019 coronavirus pandemic, which started in Wuhan, China, spread around the globe with dramatic and lethal effects. From the initial Chinese epicenter, the European diaspora taxed the resources of several countries and especially those of Italy, which was forced into a complete social and economic shutdown. Infection by droplets contaminating hands and surfaces represents the main vehicle of diffusion of the virus. The common and strong efforts to contain the pandemic have relevant effects on the management of samples from histopathology laboratories. The current commentary reports and focuses on the protocols and guidelines in use at a large tertiary Italian hospital that accordingly are proposed for adoption in Italian laboratories as a potential model for national guidelines for the coronavirus emergency.


Assuntos
Contenção de Riscos Biológicos/métodos , Infecções por Coronavirus/patologia , Técnicas Citológicas/métodos , Transmissão de Doença Infecciosa do Paciente para o Profissional/prevenção & controle , Pneumonia Viral/patologia , Contenção de Riscos Biológicos/normas , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Técnicas Citológicas/normas , Humanos , Controle de Infecções/métodos , Controle de Infecções/normas , Itália , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia
11.
Rev. cuba. med. trop ; 72(1): e500, ene.-abr. 2020. tab, graf
Artigo em Espanhol | LILACS, CUMED | ID: biblio-1126700

RESUMO

Introducción: Existen pocos estudios sobre la circulación del virus del papiloma humano en mujeres ecuatorianas, particularmente residentes en el Cantón Cañar. Objetivo: Determinar la circulación del virus del papiloma humano, las alteraciones en la citología cérvico-vaginal de mujeres cañaríes y el comportamiento de algunas variables sociodemográficas y clínico-epidemiológicas. Métodos: Estudio analítico de corte transversal desde julio 2017-septiembre 2018. Se colectaron células cervicouterinas de 100 mujeres entre 15 y 55 años de edad para determinar la infección viral y alteraciones citológicas. Se investigó la asociación entre variables sociodemográficas y clínico-epidemiológicas con la infección viral. Resultados: El 51 por ciento (51/100) de las mujeres examinadas resultó positivo al virus, con predominio de los genotipos oncogénicos. El genotipo 31 fue el más frecuente (56,9 por ciento; 29/51), seguido por el genotipo 58 (43,1 por ciento; 22/51). Las mujeres mayores de 50 años, tenían una probabilidad menor de estar infectadas (3,9 por ciento; 2/51). La probabilidad de infección fue mayor en mujeres solteras, con antecedentes de infecciones de transmisión sexual, que padecían procesos cervicales inflamatorios, y en las fumadoras. La infección con genotipo 66 estuvo asociada al uso de anticonceptivos hormonales (53,3 por ciento; 8/15); p= 0,045, RP= 3,08 IC95 por ciento (1,00-9,46). Se obtuvo el 97 por ciento de citologías negativas para malignidad; no se diagnosticaron casos con lesiones de alto grado. Conclusiones: La elevada prevalencia de infección con genotipos oncogénicos en contraste con la baja frecuencia de citologías positivas, indica la necesidad de implementar programas eficientes para la detección precoz del cáncer cervicouterino en la población del Cañar y divulgar campañas de educación sexual y reproductiva(AU)


Introduction: Few studies are available about the circulation of human papillomavirus among Ecuadorian women, particularly those from Cañar Canton. Objectives: Determine the circulation of human papillomavirus, alterations in the cervical-vaginal cytology of women from Cañar Canton, and the behavior of some sociodemographic and clinical-epidemiological variables. Methods: An analytical cross-sectional study was conducted from July 2017 to September 2018. Cervical cells were collected from 100 women aged 15-55 years to determine viral infection and cytological alterations. An analysis was performed of the relationship of sociodemographic and clinical-epidemiological variables to viral infection. Results: Of the women examined, 51 percent; (51/100) tested positive for the virus, with a predominance of oncogenic genotypes. Genotype 31 was the most common (56.9 percent;; 29/51), followed by genotype 58 (43.1 percent; 22/51). Women aged over 50 years had a lesser probability of being infected (3.9 percent;; 2/51). Infection probability was greater among single women, with a history of sexually transmitted infections, who suffered from inflammatory cervical processes, and smokers. Infection by genotype 66 was associated to the use of hormonal contraceptives (53.3 percent;; 8/15); p= 0.045, PR= 3.08 CI95 percent; (1.00-9.46). Of the sample cytologies, 97 percent; were negative for malignancy; no case was diagnosed of high-grade lesions. Conclusions: The high prevalence of infection by oncogenic genotypes, as opposed to the low frequency of positive cytologies, points to the need to implement efficient programs aimed at early detection of cervical cancer in the population of Cañar Canton, as well as sexual and reproductive education campaigns(AU)


Assuntos
Humanos , Feminino , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/epidemiologia , Estudos Transversais , Técnicas Citológicas/métodos , Equador , Genótipo
12.
Nat Protoc ; 15(4): 1560-1583, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231324

RESUMO

The human germ-cell lineage originates as human primordial germ cells (hPGCs). hPGCs undergo genome-wide epigenetic reprogramming and differentiate into oogonia or gonocytes, precursors for oocytes or spermatogonia, respectively. Here, we describe a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into oogonia in vitro. hiPSCs are induced into incipient mesoderm-like cells (iMeLCs) using activin A and a WNT pathway agonist. iMeLCs, or, alternatively, hPSCs cultured with divergent signaling inhibitors, are induced into hPGC-like cells (hPGCLCs) in floating aggregates by cytokines including bone morphogenic protein 4. hPGCLCs are aggregated with mouse embryonic ovarian somatic cells to form xenogeneic reconstituted ovaries, which are cultured under an air-liquid interface condition for ~4 months for hPGCLCs to differentiate into oogonia and immediate precursory states for oocytes. To date, this is the only approach that generates oogonia from hPGCLCs. The protocol is suitable for investigating the mechanisms of hPGC specification and epigenetic reprogramming.


Assuntos
Diferenciação Celular/fisiologia , Técnicas Citológicas/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Oogônios/citologia , Animais , Células Cultivadas , Feminino , Células Germinativas/citologia , Humanos , Mesoderma/citologia , Camundongos
13.
J Vis Exp ; (158)2020 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-32310229

RESUMO

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.


Assuntos
Movimento Celular , Técnicas Citológicas/métodos , Linhagem Celular Tumoral , Proliferação de Células , Impedância Elétrica , Glioblastoma/patologia , Humanos , Invasividade Neoplásica , Transdução de Sinais , Fatores de Tempo
16.
Cancer Cytopathol ; 128(5): 317-320, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32259373

RESUMO

The 2019 coronavirus pandemic, which started in Wuhan, China, spread around the globe with dramatic and lethal effects. From the initial Chinese epicenter, the European diaspora taxed the resources of several countries and especially those of Italy, which was forced into a complete social and economic shutdown. Infection by droplets contaminating hands and surfaces represents the main vehicle of diffusion of the virus. The common and strong efforts to contain the pandemic have relevant effects on the management of samples from histopathology laboratories. The current commentary reports and focuses on the protocols and guidelines in use at a large tertiary Italian hospital that accordingly are proposed for adoption in Italian laboratories as a potential model for national guidelines for the coronavirus emergency.


Assuntos
Contenção de Riscos Biológicos/métodos , Infecções por Coronavirus/patologia , Técnicas Citológicas/métodos , Transmissão de Doença Infecciosa do Paciente para o Profissional/prevenção & controle , Pneumonia Viral/patologia , Contenção de Riscos Biológicos/normas , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Técnicas Citológicas/normas , Humanos , Controle de Infecções/métodos , Controle de Infecções/normas , Itália , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/transmissão , Pneumonia Viral/virologia
18.
Micron ; 133: 102863, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234685

RESUMO

For diagnosing and monitoring the progress of cancer, detection and quantification of tumor cells is utmost important. Beside standard bench top instruments, several biochip-based methods have been developed for this purpose. Our biochip design incorporates micron size immunomagnetic beads together with micropad arrays, thus requires automated detection and quantification of not only cells but also the micropads and the immunomagnetic beads. The main purpose of the biochip is to capture target cells having different antigens simultaneously. In this proposed study, a digital image processing-based method to quantify the leukemia cells, immunomagnetic beads and micropads was developed as a readout method for the biochip. Color, size-based object detection and object segmentation methods were implemented to detect structures in the images acquired from the biochip by a bright field optical microscope. It has been shown that manual counting and flow cytometry results are in good agreement with the developed automated counting. Average precision is 85 % and average error rate is 13 % for all images of patient samples, average precision is 99 % and average error rate is 1% for cell culture images. With the optimized micropad size, proposed method can reach up to 95 % precision rate for patient samples with an execution time of 90 s per image.


Assuntos
Automação , Técnicas Citológicas/métodos , Processamento de Imagem Assistida por Computador/métodos , Leucemia/diagnóstico , Análise Serial de Proteínas , Humanos , Separação Imunomagnética
19.
PLoS One ; 15(3): e0230001, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32155214

RESUMO

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a valuable resource for cardiac therapeutic development; however, generation of these cells in large numbers and high purity is a limitation in widespread adoption. Here, design of experiments (DOE) is used to investigate the cardiac differentiation space of three hiPSC lines when varying CHIR99027 concentration and cell seeding density, and a novel image analysis is developed to evaluate plate coverage when initiating differentiation. Metabolic selection via lactate purifies hiPSC-cardiomyocyte populations, and the bioenergetic phenotype and engineered tissue mechanics of purified and unpurified hiPSC-cardiomyocytes are compared. Findings demonstrate that when initiating differentiation one day after hiPSC plating, low (3 µM) Chiron and 72 x 103 cells/cm2 seeding density result in peak cardiac purity (50-90%) for all three hiPSC lines. Our results confirm that metabolic selection with lactate shifts hiPSC-cardiomyocyte metabolism towards oxidative phosphorylation, but this more "mature" metabolic phenotype does not by itself result in a more mature contractile phenotype in engineered cardiac tissues at one week of culture in 3D tissues. This study provides widely adaptable methods including novel image analysis code and parameters for refining hiPSC-cardiomyocyte differentiation and describes the practical implications of metabolic selection of cardiomyocytes for downstream tissue engineering applications.


Assuntos
Diferenciação Celular , Técnicas Citológicas/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Adulto , Linhagem Celular , Humanos , Miócitos Cardíacos/metabolismo , Fenótipo
20.
Methods Cell Biol ; 156: 185-203, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32222219

RESUMO

Cell migration is involved in key phenomena in biology, ranging from development to cancer. Fibroblasts move between organs in 3D polymeric networks. So far, motile cells were mainly tracked in vitro on Petri dishes or on coverslips, i.e., 2D flat surfaces, which made the extrapolation to 3D physiological environments difficult. We therefore prepared 3D Cell Derived Matrices (CDM) with specific characteristics with the goal of extracting the main readouts required to measure and characterize cell motion: cell specific matrix deformation through the tracking of fluorescent fibronectin within CDM, focal contacts as the cell anchor and acto-myosin cytoskeleton which applies cellular forces. We report our method for generating this assay of physiological-like gel with relevant readouts together with its potential impact in explaining cell motility in vivo.


Assuntos
Movimento Celular , Técnicas Citológicas/métodos , Matriz Extracelular/metabolismo , Imageamento Tridimensional , Algoritmos , Animais , Fluorescência , Células HeLa , Humanos , Camundongos , Células NIH 3T3
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