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1.
Nat Commun ; 11(1): 4851, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32978386

RESUMO

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.


Assuntos
Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Ligantes , Engenharia Metabólica/métodos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Cristalografia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Lisina/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Termodinâmica
2.
PLoS One ; 15(8): e0237748, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866195

RESUMO

Soil microbiota are considered a source of undiscovered bioactive compounds, yet cultivation of most bacteria within a sample remains generally unsuccessful. Two main reasons behind the unculturability of bacteria are the presence of cells in a viable but not culturable state (such as dormant cells) and the failure to provide the necessary growth requirements in vitro (leading to the classification of some bacterial taxa as yet-to-be-cultured). The present work focuses on the development of a single procedure that helps distinguish between both phenomena of unculturability based on viability staining coupled with flow cytometry and fluorescence-activated cell sorting. In the selected soil sample, the success rate of cultured bacteria was doubled by selecting viable and metabolically active bacteria. It was determined that most of the uncultured fraction was not dormant or dead but likely required different growth conditions. It was also determined that the staining process introduced changes in the taxonomic composition of the outgrown bacterial biomass, which should be considered for further developments. This research shows the potential of flow cytometry and fluorescence-activated cell sorting applied to soil samples to improve the success rate of bacterial cultivation by estimating the proportion of dormant and yet-to-be-cultured bacteria and by directly excluding dormant cells from being inoculated into growth media.


Assuntos
Bactérias/crescimento & desenvolvimento , Técnicas Microbiológicas/métodos , Microbiota/fisiologia , Microbiologia do Solo , Bactérias/química , Bactérias/genética , Biomassa , Separação Celular/métodos , Meios de Cultura , DNA Bacteriano/isolamento & purificação , Estudos de Viabilidade , Citometria de Fluxo/métodos , Corantes Fluorescentes/química , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Coloração e Rotulagem/métodos
3.
PLoS One ; 15(8): e0237155, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32866200

RESUMO

BACKGROUND: Stringent complete response (sCR) is used as a deeper response category than complete response (CR) in multiple myeloma (MM) but may be of limited value in the era of minimal residual disease (MRD) testing. METHODS: Here, we used 4-colour multiparametric flow cytometry (MFC) or next-generation sequencing (NGS) of immunoglobulin genes to analyse and compare the prognostic impact of sCR and MRD monitoring. We included 193 treated patients in two institutions achieving CR, for which both bone marrow aspirates and biopsies were available. RESULTS: We found that neither the serum free light chain ratio, clonality by immunohistochemistry (IHC) nor plasma cell bone marrow infiltration identified CR patients at distinct risk. Patients with sCR had slightly longer progression-free survival. Nevertheless, persistent clonal bone marrow disease was detectable using MFC or NGS and was associated with significantly inferior outcomes compared with MRD-negative cases. CONCLUSION: Our results confirm that sCR does not predict a different outcome and indicate that more sensitive techniques are able to identify patients with differing prognoses. We suggest that MRD categories should be implemented over sCR for the future classification of MM responses.


Assuntos
Mieloma Múltiplo/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/patologia , Confiabilidade dos Dados , Feminino , Citometria de Fluxo/métodos , Seguimentos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Cadeias Leves de Imunoglobulina/sangue , Cadeias Leves de Imunoglobulina/genética , Masculino , Pessoa de Meia-Idade , Neoplasia Residual , Plasmócitos/imunologia , Prognóstico , Intervalo Livre de Progressão , Estudos Retrospectivos
4.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32817357

RESUMO

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , França/epidemiologia , Voluntários Saudáveis , Humanos , Imunoprecipitação/métodos , Luciferases , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Médica Translacional , Adulto Jovem
5.
Sci Immunol ; 5(50)2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826343

RESUMO

Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.


Assuntos
Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Células Matadoras Naturais/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Índice de Gravidade de Doença , Imunidade Adaptativa , Antígeno CD56/metabolismo , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Citometria de Fluxo/métodos , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , Fenótipo , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Reação em Cadeia da Polimerase , Estudos Prospectivos , Mapas de Interação de Proteínas/imunologia , Receptores KIR/metabolismo , Testes Sorológicos , Suécia/epidemiologia
6.
Protein Cell ; 11(10): 740-770, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32780218

RESUMO

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.


Assuntos
Envelhecimento/imunologia , Betacoronavirus , Infecções por Coronavirus/imunologia , Sistema Imunitário/imunologia , Pandemias , Pneumonia Viral/imunologia , Análise de Célula Única , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Linfócitos T CD4-Positivos/metabolismo , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/imunologia , Citocinas/biossíntese , Citocinas/genética , Suscetibilidade a Doenças , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Rearranjo Gênico , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/crescimento & desenvolvimento , Imunocompetência/genética , Inflamação/genética , Inflamação/imunologia , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Análise de Sequência de RNA , Transcriptoma , Adulto Jovem
7.
Protein Cell ; 11(10): 740-770, 2020 10.
Artigo em Inglês | MEDLINE | ID: covidwho-709445

RESUMO

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.


Assuntos
Envelhecimento/imunologia , Betacoronavirus , Infecções por Coronavirus/imunologia , Sistema Imunitário/imunologia , Pandemias , Pneumonia Viral/imunologia , Análise de Célula Única , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/genética , Linfócitos T CD4-Positivos/metabolismo , Linhagem da Célula , Montagem e Desmontagem da Cromatina , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/imunologia , Citocinas/biossíntese , Citocinas/genética , Suscetibilidade a Doenças , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Rearranjo Gênico , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/crescimento & desenvolvimento , Imunocompetência/genética , Inflamação/genética , Inflamação/imunologia , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Análise de Sequência de RNA , Transcriptoma , Adulto Jovem
8.
Sci Immunol ; 5(50)2020 08 21.
Artigo em Inglês | MEDLINE | ID: covidwho-725061

RESUMO

Understanding innate immune responses in COVID-19 is important to decipher mechanisms of host responses and interpret disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections but might also contribute to immunopathology. Using 28-color flow cytometry, we here reveal strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients. This pattern was mirrored in scRNA-seq signatures of NK cells in bronchoalveolar lavage from COVID-19 patients. Unsupervised high-dimensional analysis of peripheral blood NK cells furthermore identified distinct NK cell immunotypes that were linked to disease severity. Hallmarks of these immunotypes were high expression of perforin, NKG2C, and Ksp37, reflecting increased presence of adaptive NK cells in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed across COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This study provides a detailed map of the NK cell activation landscape in COVID-19 disease.


Assuntos
Betacoronavirus/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Células Matadoras Naturais/imunologia , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Índice de Gravidade de Doença , Imunidade Adaptativa , Antígeno CD56/metabolismo , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Citometria de Fluxo/métodos , Humanos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Pandemias , Fenótipo , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Reação em Cadeia da Polimerase , Estudos Prospectivos , Mapas de Interação de Proteínas/imunologia , Receptores KIR/metabolismo , Testes Sorológicos , Suécia/epidemiologia
9.
Sci Transl Med ; 12(559)2020 09 02.
Artigo em Inglês | MEDLINE | ID: covidwho-724557

RESUMO

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , França/epidemiologia , Voluntários Saudáveis , Humanos , Imunoprecipitação/métodos , Luciferases , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Estudos Soroepidemiológicos , Glicoproteína da Espícula de Coronavírus/imunologia , Pesquisa Médica Translacional , Adulto Jovem
10.
PLoS One ; 15(8): e0236439, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813744

RESUMO

Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.


Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/isolamento & purificação , Vesículas Extracelulares/genética , Proteoma/genética , Adulto , Plaquetas/química , Proteínas Sanguíneas/genética , Feminino , Citometria de Fluxo/métodos , Voluntários Saudáveis , Humanos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade
11.
PLoS One ; 15(8): e0237157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760102

RESUMO

Ants (Formicidae) present considerable diversity in chromosome numbers, which vary from n = 1 to n = 60, although this variation is not proportional to that in genome size, for which estimates range from 0.18 pg to 0.77 pg. Intraspecific variation in the chromosome number and karyotype structure has been reported among species, although the variation among populations of the same species has received much less attention, and there are few data on genome size. Here, we studied the karyotype length and genome size of different populations of the fungus-farming ants Mycetophylax conformis (Mayr, 1884) and Mycetophylax morschi (Emery, 1888). We also provide remarks on procedure for the estimation of ant genome size by Flow Cytometry (FCM) analysis. Chromosome number and morphology did not vary among the populations of M. conformis or the cytotypes of M. morschi, but karyotype length and genome size were significantly distinct among the populations of these ants. Our results on the variation in karyotype length and genome size among M. morschi and M. conformis populations reveal considerable diversity that would be largely overlooked by more traditional descriptions of karyotypes, which were also supported by the estimates of genome size obtained using flow cytometry. Changes in the amount of DNA reflect variation in the fine structure of the chromosomes, which may represent the first steps of karyotype evolution and may occur previously to any changes in the chromosome number.


Assuntos
Formigas/genética , Cromossomos de Insetos/genética , Variação Genética , Genoma de Inseto , Cariótipo , Animais , Citometria de Fluxo/métodos , Cariotipagem/métodos
12.
Medicine (Baltimore) ; 99(27): e20726, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32629647

RESUMO

INTRODUCTION: Scleromyxedema (rare cutaneous mucinosis), is characterized by the formation of lichenoid papules and presence of Serum monoclonal IgG in most cases, or all; after repeated testing. PATIENT CONCERNS: The patient is a 51-year-old male presented with thick, disfiguring elephant-like erythematous skin folds over the forehead, papular shiny eruptions over ears and trunk and waxy erythematous papules over arms and hands without dysphagia or respiratory or neurologic symptoms DIAGNOSIS: : Skin biopsy from right arm was consistent with scleromyxedema. Serum cryoglobulin was reported negative. Complete blood count and routine blood biochemistry were normal. Thyroid function tests were normal. Serum protein electrophoresis and immunofixation showed monoclonal band of 14.5 g/L typed as IgG lambda. INTERVENTIONS: Our patient was refractory to lenalidomide however improved clinically on immunoglobulins infusions on monthly basis without change in the MGUS level. OUTCOMES: NGF analysis revealed approximately 0.25% Lambda monotypic plasma cells in the bone marrow expressing CD38, CD138, and CD27 with aberrant expression of CD56 and were negative for CD45, CD19, CD117, and CD81. We also detected 0.002% circulating plasma cells (PCs) in peripheral blood. CONCLUSION: The immunophenotype of circulating tumor cells (CTCs) remain close to the malignant PCs phenotype in the BM. Hence, we report NGF approach as a novel diagnostic tool for highly sensitive MRD detection in plasma cell dyscrasias including scleromyxedema.


Assuntos
Citometria de Fluxo/métodos , Células Neoplásicas Circulantes/patologia , Escleromixedema/patologia , Orelha Externa/patologia , Testa/patologia , Humanos , Imunoglobulina G/sangue , Imunoglobulinas Intravenosas/administração & dosagem , Imunoglobulinas Intravenosas/uso terapêutico , Masculino , Pessoa de Meia-Idade , Paraproteinemias/imunologia , Paraproteinemias/patologia , Escleromixedema/terapia , Pele/patologia
13.
J Vis Exp ; (161)2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32716395

RESUMO

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.


Assuntos
Autofagia/fisiologia , Bioensaio/métodos , Condrócitos/patologia , Citometria de Fluxo/métodos , Osteoartrite/patologia , Linhagem Celular Transformada , Condrócitos/efeitos dos fármacos , Descoberta de Drogas/métodos , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/farmacologia , Proteínas Associadas aos Microtúbulos/uso terapêutico , Osteoartrite/tratamento farmacológico , Osteoartrite/fisiopatologia
14.
Scand J Immunol ; 92(3): e12924, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32602962

RESUMO

Magnetic-activated cell sorting (MACS) using magnetic nanoparticles coated with specific antibodies is commonly used in immunology research. For in vitro isolation purposes, it is important to know to what extent the magnetic properties remain present in the isolated cell populations and whether it has consequences for sequential isolations. We hypothesized that only upon cell division, cells will lose their magnetic properties via dilution of the particles in/on their daughter cells. We analysed residual magnetic properties of cells that divided vs cells that did not divide after magnetic bead-based cell separation. As a model, we isolated T cells using beads targeting the non-modulating surface molecule CD45RO. Cells were labelled with the cell division tracking dye PKH and cultured under different conditions to induce variable degrees of cell division. We demonstrate that T cells that underwent no, or only minimal, cell divisions after MACS retained magnetic properties for up to at least 2 weeks of in vitro culture. The presence of nanoparticles was detected on their cell surface and intracellularly using Labeling Check reagent. These results have important consequences for procedures requiring repetitive isolation rounds after in vitro culture.


Assuntos
Separação Imunomagnética/métodos , Linfócitos T/citologia , Linfócitos T/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Proliferação de Células , Citometria de Fluxo/métodos , Humanos , Memória Imunológica , Ativação Linfocitária/imunologia , Linfócitos T/imunologia
15.
Exp Parasitol ; 216: 107940, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32562606

RESUMO

Therapeutic options for the treatment of leishmaniasis are insufficient and need improvements owing to their low efficiency and high toxicity as well as the emergence of resistant strains. The limited number of new drugs for neglected diseases and lack of innovation in your development are still challenges. In this context, the process of discovery and development of biological assays play a pivotal role for the identification of bioactive compounds. The assays currently used for screening of drugs with cytotoxic activity against Leishmania parasites, include different processes that utilize intact parasite (free or intracellular) or specific enzymes of metabolism as a target cell. These assays allow the screening of large numbers of samples followed by more detailed secondary confirmatory assays to confirm the observed activity and assess their toxicity. In the present study, we described the development of a new functional and more complete assay that enables simultaneous assessment of potential anti-Leishmania compounds through evaluation of internalization of fluorescein-labeled L. braziliensis promastigotes by human peripheral blood monocytes and their cytotoxicity by flow cytometry. We standardized the conditions for parasite labeling to achieve better phagocytosis analysis by setting the ratio of number of parasites per cell as 1 to 2, at incubation time of 6h. The cytotoxicity assessment was performed by the quantification of cells undergoing early/late apoptosis and necrosis using a double labelling platform employing 7AAD for late apoptosis and necrosis analysis and Annexin-V for early apoptosis evaluation. Hemolysis analysis was an additional parameter to test cytotoxicity. Two drugs used on clinic (Amphotericin B and Glucantime®) were used to validate the proposed methodology, and the assay was able to detect their known leishmanicidal activity and immunotoxicity properties. This new predictive assay will contribute to the development of translational medicine strategies in drug discovery for neglected diseases such as leishmaniasis.


Assuntos
Alternativas aos Testes com Animais/métodos , Antiprotozoários/toxicidade , Citometria de Fluxo/métodos , Leishmania/efeitos dos fármacos , Doenças Negligenciadas/tratamento farmacológico , Adulto , Anfotericina B/farmacologia , Anfotericina B/toxicidade , Animais , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Leishmania braziliensis/efeitos dos fármacos , Leishmaniose/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Leucócitos/parasitologia , Antimoniato de Meglumina/farmacologia , Antimoniato de Meglumina/uso terapêutico , Antimoniato de Meglumina/toxicidade , Microscopia Confocal , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , Fatores de Tempo , Adulto Jovem
16.
PLoS One ; 15(6): e0233961, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32479512

RESUMO

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.


Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Biblioteca de Peptídeos , Alanina/genética , Alanina/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas de Visualização da Superfície Celular/economia , Análise Custo-Benefício , Citometria de Fluxo/economia , Citometria de Fluxo/métodos , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Mutação , Ligação Proteica/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
PLoS One ; 15(6): e0233443, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497056

RESUMO

Large (> 1 µm) tumor-derived extracellular vesicles (tdEVs) enriched from the cell fraction of centrifuged whole blood are prognostic in metastatic castration-resistant prostate cancer (mCRPC) patients. However, the highest concentration of tdEVs is expected in the cell-free plasma fraction. In this pilot study, we determine whether mCRPC patients can be discriminated from healthy controls based on detection of tdEVs (< 1µm, EpCAM+) and/or other EVs, in cell-free plasma and/or urine. The presence of marker+ EVs in plasma and urine samples from mCRPC patients (n = 5) and healthy controls (n = 5) was determined by flow cytometry (FCM) and surface plasmon resonance imaging (SPRi) using an antibody panel and lactadherin. For FCM, the concentrations of marker positive (+) particles and EVs (refractive index <1.42) were determined. Only the lactadherin+ particle and EV concentration in plasma measured by FCM differed significantly between patients and controls (p = 0.017). All other markers did not result in signals exceeding the background on both FCM and SPRi, or did not differ significantly between patients and controls. In conclusion, no difference was found between patients and controls based on the detection of tdEVs. For FCM, the measured sample volumes are too small to detect tdEVs. For SPRi, the concentration of tdEVs is probably too low to be detected. Thus, to detect tdEVs in cell-free plasma and/or urine, EV enrichment and/or concentration is required. Furthermore, we recommend testing other markers and/or a combination of markers to discriminate mCRPC patients from healthy controls.


Assuntos
Adenocarcinoma/secundário , Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Neoplasias de Próstata Resistentes à Castração/sangue , Neoplasias de Próstata Resistentes à Castração/urina , Ressonância de Plasmônio de Superfície/métodos , Adenocarcinoma/sangue , Adenocarcinoma/urina , Idoso , Idoso de 80 Anos ou mais , Antígenos de Superfície/sangue , Biomarcadores Tumorais , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Vesículas Extracelulares/química , Humanos , Masculino , Proteínas do Leite/sangue , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/urina , Projetos Piloto , Neoplasias de Próstata Resistentes à Castração/patologia
18.
PLoS One ; 15(6): e0234125, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492063

RESUMO

Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea aerocolonigenes were investigated by microscopy and flow cytometry. Both methods were compared regarding their applicability. To achieve different morphologies, a cultivation with glass bead addition (Ø = 969 µm, 100 g L-1) was compared to an unsupplemented cultivation. This led to two different macro-morphologies. Furthermore, glass bead addition increased rebeccamycin titers after 10 days of cultivation (95 mg L-1 with glass beads, 38 mg L-1 without glass beads). Macro-morphology and viability were investigated through microscopy and flow cytometry. For viability assessment fluorescent staining was used additionally. Smaller, more regular pellets were found for glass bead addition. Pellet diameters resulting from microscopy followed by image analysis were 172 µm without and 106 µm with glass beads, diameters from flow cytometry were 170 and 100 µm, respectively. These results show excellent agreement of both methods, each considering several thousand pellets. Furthermore, the pellet viability obtained from both methods suggested an enhanced metabolic activity in glass bead treated pellets during the exponential production phase. However, total viability values differ for flow cytometry (0.32 without and 0.41 with glass beads) and confocal laser scanning microscopy of single stained pellet slices (life ratio in production phase of 0.10 without and 0.22 with glass beads), which is probably caused by the different numbers of investigated pellets. In confocal laser scanning microscopy only one pellet per sample could be investigated while flow cytometry considered at least 50 pellets per sample, resulting in an increased statistical reliability.


Assuntos
Actinomycetales/fisiologia , Citometria de Fluxo/métodos , Microscopia/métodos , Actinomycetales/citologia , Carbazóis/análise , Cromatografia Líquida de Alta Pressão , Processamento de Imagem Assistida por Computador , Microscopia Confocal
19.
Nucleic Acids Res ; 48(15): e86, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32544240

RESUMO

Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.


Assuntos
DNA/isolamento & purificação , Citometria de Fluxo/métodos , Imagem Individual de Molécula/métodos , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Humanos , Hibridização In Situ/métodos , Linfócitos/química , Reação em Cadeia da Polimerase/métodos
20.
J Vis Exp ; (159)2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32510500

RESUMO

Increasing evidence supports the hypothesis that neuro-immune interactions impact nervous system function in both homeostatic and pathologic conditions. A well-studied function of major histocompatibility complex class I (MHCI) is the presentation of cell-derived peptides to the adaptive immune system, particularly in response to infection. More recently it has been shown that the expression of MHCI molecules on neurons can modulate activity-dependent changes in the synaptic connectivity during normal development and neurologic disorders. The importance of these functions to the brain health supports the need for a sensitive assay that readily detects MHCI expression on neurons. Here we describe a method for primary culture of murine hippocampal neurons and then assessment of MHCI expression by flow cytometric analysis. Murine hippocampus is microdissected from prenatal mouse pups at the embryonic day 18. Tissue is dissociated into a single cell suspension using enzymatic and mechanical techniques, then cultured in a serum-free media that limits growth of non-neuronal cells. After 7 days in vitro, MHCI expression is stimulated by treating cultured cells pharmacologically with beta interferon. MHCI molecules are labeled in situ with a fluorescently tagged antibody, then cells are non-enzymatically dissociated into a single cell suspension. To confirm the neuronal identity, cells are fixed with paraformaldehyde, permeabilized, and labeled with a fluorescently tagged antibody that recognizes neuronal nuclear antigen NeuN. MHCI expression is then quantified on neurons by flow cytometric analysis. Neuronal cultures can easily be manipulated by either genetic modifications or pharmacologic interventions to test specific hypotheses. With slight modifications, these methods can be used to culture other neuronal populations or to assess expression of other proteins of interest.


Assuntos
Citometria de Fluxo/métodos , Hipocampo/citologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Feminino , Hipocampo/embriologia , Camundongos Endogâmicos C57BL , Neurônios/citologia
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