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1.
BMC Genomics ; 22(Suppl 5): 518, 2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34789161

RESUMO

BACKGROUND: All diseases containing genetic material undergo genetic evolution and give rise to heterogeneity including cancer and infection. Although these illnesses are biologically very different, the ability for phylogenetic retrodiction based on the genomic reads is common between them and thus tree-based principles and assumptions are shared. Just as the different frequencies of tumor genomic variants presupposes the existence of multiple tumor clones and provides a handle to computationally infer them, we postulate that the different variant frequencies in viral reads offers the means to infer multiple co-infecting sublineages. RESULTS: We present a common methodological framework to infer the phylogenomics from genomic data, be it reads of SARS-CoV-2 of multiple COVID-19 patients or bulk DNAseq of the tumor of a cancer patient. We describe the Concerti computational framework for inferring phylogenies in each of the two scenarios.To demonstrate the accuracy of the method, we reproduce some known results in both scenarios. We also make some additional discoveries. CONCLUSIONS: Concerti successfully extracts and integrates information from multi-point samples, enabling the discovery of clinically plausible phylogenetic trees that capture the heterogeneity known to exist both spatially and temporally. These models can have direct therapeutic implications by highlighting "birth" of clones that may harbor resistance mechanisms to treatment, "death" of subclones with drug targets, and acquisition of functionally pertinent mutations in clones that may have seemed clinically irrelevant. Specifically in this paper we uncover new potential parallel mutations in the evolution of the SARS-CoV-2 virus. In the context of cancer, we identify new clones harboring resistant mutations to therapy.


Assuntos
COVID-19 , Neoplasias , Células Clonais , Humanos , Mutação , Neoplasias/genética , Filogenia , SARS-CoV-2
2.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639148

RESUMO

The stemness in keratinocyte stem cells (KSCs) is determined by their gene expression patterns. KSCs are crucial in maintaining epidermal homeostasis and wound repair and are widely used candidates for therapeutic applications. Although several studies have reported their positive identifiers, unique biomarkers for KSCs remain elusive. Here, we aim to identify potential candidate stem cell markers. Human epidermal keratinocytes (HEKs) from neonatal foreskin tissues were isolated and cultured. Single-cell clonal analysis identified and characterized three types of cells: KSCs (holoclones), transient amplifying cells (TACs; meroclones), and differentiated cells (DSCs; paraclones). The clonogenic potential of KSCs demonstrated the highest proliferation potential of KSCs, followed by TACs and DSCs, respectively. Whole-transcriptome analysis using microarray technology unraveled the molecular signatures of these cells. These results were validated by quantitative real-time polymerase chain reaction and flow cytometry analysis. A total of 301 signature upregulated and 149 downregulated differentially expressed genes (DEGs) were identified in the KSCs, compared to TACs and DSCs. Furthermore, DEG analyses revealed new sets of genes related to cell proliferation, cell adhesion, surface makers, and regulatory factors. In conclusion, this study provides a useful source of information for the identification of potential SC-specific candidate markers.


Assuntos
Biomarcadores/metabolismo , Células Clonais/metabolismo , Células Epidérmicas/metabolismo , Regulação da Expressão Gênica , Queratinócitos/metabolismo , Células-Tronco/metabolismo , Diferenciação Celular , Células Cultivadas , Células Clonais/citologia , Células Epidérmicas/citologia , Perfilação da Expressão Gênica , Humanos , Queratinócitos/citologia , Células-Tronco/citologia
3.
J R Soc Interface ; 18(183): 20210607, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34637643

RESUMO

During ageing, normal epithelial tissues progressively accumulate clones carrying mutations that increase mutant cell fitness above that of wild-type cells. Such mutants spread widely through the tissues, yet despite this cellular homeostasis and functional integrity of the epithelia are maintained. Two of the genes most commonly mutated in human skin and oesophagus are p53 and Notch1, both of which are also recurrently mutated in cancers of these tissues. From observations taken in human and mouse epithelia, we find that clones carrying p53 and Notch pathway mutations have different clone dynamics which can be explained by their different responses to local cell crowding. p53 mutant clone growth in mouse epidermis approximates a logistic curve, but feedbacks responding to local crowding are required to maintain tissue homeostasis. We go on to show that the observed ability of Notch pathway mutant cells to displace the wild-type population in the mouse oesophageal epithelium reflects a local density feedback that affects both mutant and wild-type cells equally. We then show how these distinct feedbacks are consistent with the distribution of mutations observed in human datasets and are suggestive of a putative mechanism to constrain these cancer-associated mutants.


Assuntos
Epitélio , Receptor Notch1 , Proteína Supressora de Tumor p53 , Animais , Carcinoma de Células Escamosas , Células Clonais , Camundongos , Mutação , Receptor Notch1/genética , Proteína Supressora de Tumor p53/genética
4.
BMC Res Notes ; 14(1): 400, 2021 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-34715916

RESUMO

OBJECTIVE: The clone devaluation is a phenomenon reported by the latest paper in which eeriness is evoked when people observe individuals with the same face (clone faces) compared to those with different faces. There are two possibilities that explain the clone devaluation effect. One is that the same facial features that clone faces have (duplication of facial features) induce the clone devaluation effect. The other possibility is that duplication of identities between people with clone faces is important for the clone devaluation effect. We thus conducted an experiment to investigate whether the duplication of identities or of facial features induces the clone devaluation effect. RESULTS: Participants evaluated eeriness of scrambled clone faces and scrambled different faces using the paired comparison method. There was only a slight difference in subjective eeriness between scrambled clone faces and scrambled different faces. Therefore, this study suggests that the duplication of local facial features does not play a key role in inducing the clone devaluation effect.


Assuntos
Face , Células Clonais , Humanos
5.
BMC Genomics ; 22(1): 697, 2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34579659

RESUMO

BACKGROUND: Nitrogen (N) is one of the main factors limiting the wood yield in poplar cultivation. Understanding the molecular mechanism of N utilization could play a guiding role in improving the nitrogen use efficiency (NUE) of poplar. RESULTS: In this study, three N-efficient genotypes (A1-A3) and three N-inefficient genotypes (C1-C3) of Populus deltoides were cultured under low N stress (5 µM NH4NO3) and normal N supply (750 µM NH4NO3). The dry matter mass, leaf morphology, and chlorophyll content of both genotypes decreased under N starvation. The low nitrogen adaptation coefficients of the leaves and stems biomass of group A were significantly higher than those of group C (p < 0.05). Interestingly, N starvation induced fine root growth in group A, but not in group C. Next, a detailed time-course analysis of enzyme activities and gene expression in leaves identified 2062 specifically differentially expressed genes (DEGs) in group A and 1118 in group C. Moreover, the sensitivity to N starvation of group A was weak, and DEGs related to hormone signal transduction and stimulus response played an important role in the low N response this group. Weighted gene co-expression network analysis identified genes related to membranes, catalytic activity, enzymatic activity, and response to stresses that might be critical for poplar's adaption to N starvation and these genes participated in the negative regulation of various biological processes. Finally, ten influential hub genes and twelve transcription factors were identified in the response to N starvation. Among them, four hub genes were related to programmed cell death and the defense response, and PodelWRKY18, with high connectivity, was involved in plant signal transduction. The expression of hub genes increased gradually with the extension of low N stress time, and the expression changes in group A were more obvious than those in group C. CONCLUSIONS: Under N starvation, group A showed stronger adaptability and better NUE than group C in terms of morphology and physiology. The discovery of hub genes and transcription factors might provide new information for the analysis of the molecular mechanism of NUE and its improvement in poplar.


Assuntos
Populus , Células Clonais/metabolismo , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Populus/genética , Populus/metabolismo , Estresse Fisiológico/genética , Áreas Alagadas
6.
Nat Commun ; 12(1): 5444, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34521850

RESUMO

Kawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although functional and phenotypic changes of immune cells have been reported, a global understanding of immune responses underlying acute KD is unclear. Here, using single-cell RNA sequencing, we profile peripheral blood mononuclear cells from seven patients with acute KD before and after intravenous immunoglobulin therapy and from three age-matched healthy controls. The most differentially expressed genes are identified in monocytes, with high expression of pro-inflammatory mediators, immunoglobulin receptors and low expression of MHC class II genes in acute KD. Single-cell RNA sequencing and flow cytometry analyses, of cells from an additional 16 KD patients, show that although the percentage of total B cells is substantially decreased after therapy, the percentage of plasma cells among the B cells is significantly increased. The percentage of CD8+ T cells is decreased in acute KD, notably effector memory CD8+ T cells compared with healthy controls. Oligoclonal expansions of both B cell receptors and T cell receptors are observed after therapy. We identify biological processes potentially underlying the changes of each cell type. The single-cell landscape of both innate and adaptive immune responses provides insights into pathogenesis and therapy of KD.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Monócitos/imunologia , Síndrome de Linfonodos Mucocutâneos/genética , Plasmócitos/imunologia , Doença Aguda , Imunidade Adaptativa/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Estudos de Casos e Controles , Proliferação de Células , Criança , Pré-Escolar , Células Clonais , Feminino , Expressão Gênica , Humanos , Imunidade Inata/efeitos dos fármacos , Imunoglobulinas Intravenosas/uso terapêutico , Imunofenotipagem , Masculino , Monócitos/efeitos dos fármacos , Monócitos/patologia , Síndrome de Linfonodos Mucocutâneos/tratamento farmacológico , Síndrome de Linfonodos Mucocutâneos/imunologia , Síndrome de Linfonodos Mucocutâneos/patologia , Plasmócitos/efeitos dos fármacos , Plasmócitos/patologia , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Análise de Sequência de RNA , Análise de Célula Única
7.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576132

RESUMO

Although anti-cancer properties of the natural compound curcumin have been reported, low absorption and rapid metabolisation limit clinical use. The present study investigated whether irradiation with visible light may enhance the inhibitory effects of low-dosed curcumin on prostate cancer cell growth, proliferation, and metastasis in vitro. DU145 and PC3 cells were incubated with low-dosed curcumin (0.1-0.4 µg/mL) and subsequently irradiated with 1.65 J/cm2 visible light for 5 min. Controls remained untreated and/or non-irradiated. Cell growth, proliferation, apoptosis, adhesion, and chemotaxis were evaluated, as was cell cycle regulating protein expression (CDK, Cyclins), and integrins of the α- and ß-family. Curcumin or light alone did not cause any significant effects on tumor growth, proliferation, or metastasis. However, curcumin combined with light irradiation significantly suppressed tumor growth, adhesion, and migration. Phosphorylation of CDK1 decreased and expression of the counter-receptors cyclin A and B was diminished. Integrin α and ß subtypes were also reduced, compared to controls. Irradiation distinctly enhances the anti-tumor potential of curcumin in vitro and may hold promise in treating prostate cancer.


Assuntos
Curcumina/farmacologia , Luz , Neoplasias da Próstata/patologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Adesão Celular/efeitos dos fármacos , Adesão Celular/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Células Clonais , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrinas/metabolismo , Masculino , Metástase Neoplásica
8.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576152

RESUMO

Aryl hydrocarbon receptor (AHR) genomic pathway has been well-characterized in a number of respiratory diseases. In addition, the cytoplasmic AHR protein may act as an adaptor of E3 ubiquitin ligase. In this study, the physiological functions of AHR that regulate cell proliferation were explored using the CRISPR/Cas9 system. The doubling-time of the AHR-KO clones of A549 and BEAS-2B was observed to be prolonged. The attenuation of proliferation potential was strongly associated with either the induction of p27Kip1 or the impairment in mitogenic signal transduction driven by the epidermal growth factor (EGF) and EGF receptor (EGFR). We found that the leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1), a repressor of EGFR, was induced in the absence of AHR in vitro and in vivo. The LRIG1 tends to degrade via a proteasome dependent manner by interacting with AHR in wild-type cells. Either LRIG1 or a disintegrin and metalloprotease 17 (ADAM17) were accumulated in AHR-defective cells, consequently accelerating the degradation of EGFR, and attenuating the response to mitogenic stimulation. We also affirmed low AHR but high LRIG1 levels in lung tissues of chronic obstructive pulmonary disease (COPD) patients. This might partially elucidate the sluggish tissue repairment and developing inflammation in COPD patients.


Assuntos
Receptores ErbB/metabolismo , Glicoproteínas de Membrana/metabolismo , Mitógenos/metabolismo , Proteólise , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Células A549 , Proteína ADAM17/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Clonais , Fator de Crescimento Epidérmico/farmacologia , Humanos , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Regulação para Cima/efeitos dos fármacos
9.
BMC Neurol ; 21(1): 352, 2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34517832

RESUMO

BACKGROUND: Rosai-Dorfman disease (RDD) is a rare, benign, idiopathic non-Langerhans cell histiocytosis. Cases of RDD in the CNS are extremely rare but lethal. RDD is thought to represent a reactive process. Recent studies proposed a subset of RDD cases that had a clonal nature. However, its clone origin is poorly understood. CASE PRESENTATION: We present a rare case of RDD in the CNS with two isolated lesions. These two lesions were removed successively after two operations. No seizure nor recurrence appears to date (2 years follow-up). Morphological and immunohistochemical profiles of these two lesions support the diagnosis of RDD. Based on the whole-exome sequencing (WES) data, we found the larger lesion has a higher tumor mutational burden (TMB) and more driver gene mutations than the smaller lesion. We also found seven common truncal mutations in these two lesions, raising the possibility that they might stem from the same ancestor clone. CONCLUSIONS: Overall, this is the first report about clonal evolution of RDD in the CNS with two isolated lesions. Our findings contribute to the pathology of RDD, and support the notion that a subset of cases with RDD is a clonal histiocytic disorder driven by genetic alterations.


Assuntos
Histiocitose Sinusal , Sistema Nervoso Central , Células Clonais , Histiocitose Sinusal/diagnóstico , Histiocitose Sinusal/genética , Humanos , Mutação/genética , Recidiva
10.
Nat Commun ; 12(1): 5330, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504093

RESUMO

Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives.


Assuntos
Alelos , Desequilíbrio Alélico , Epigênese Genética , Doenças Musculares/genética , Células-Tronco Neurais/metabolismo , Doenças Neurodegenerativas/genética , Hidrolases Anidrido Ácido/genética , Hidrolases Anidrido Ácido/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Diferenciação Celular , Quimera , Células Clonais , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Dosagem de Genes , Frequência do Gene , Loci Gênicos , Impressão Genômica , Masculino , Camundongos , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neurais/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo
11.
J Immunol ; 207(8): 2086-2095, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34551963

RESUMO

CMV is a major infectious complication following solid organ transplantation. Reactivation of CMV leads to memory inflation, a process in which CD8 T cells expand over time. Memory inflation is associated with specific changes in T cell function, including increased oligoclonality, decreased cytokine production, and terminal differentiation. To address whether memory inflation during the first year after transplantation in human subjects alters T cell differentiation and function, we employed single-cell-matched TCRαß and targeted gene expression sequencing. Expanded T cell clones exhibited a terminally differentiated, immunosenescent, and polyfunctional phenotype whereas rare clones were less differentiated. Clonal expansion occurring between pre- and 3 mo posttransplant was accompanied by enhancement of polyfunctionality. In contrast, polyfunctionality and differentiation state were largely maintained between 3 and 12 mo posttransplant. Highly expanded clones had a higher degree of polyfunctionality than rare clones. Thus, CMV-responsive CD8 T cells differentiated during the pre- to posttransplant period then maintained their differentiation state and functional capacity despite posttransplant clonal expansion.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Transplante de Coração , Transplante de Rim , Adulto , Idoso , Antígenos Virais/imunologia , Diferenciação Celular , Proliferação de Células , Células Clonais , Feminino , Humanos , Memória Imunológica , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Análise de Célula Única
12.
J Immunol ; 207(8): 2077-2085, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34551964

RESUMO

CMV infection is a significant complication after solid organ transplantation. We used single cell TCR αß sequencing to determine how memory inflation impacts clonality and diversity of the CMV-responsive CD8 and CD4 T cell repertoire in the first year after transplantation in human subjects. We observed CD8 T cell inflation but no changes in clonal diversity, indicating homeostatic stability in clones. In contrast, the CD4 repertoire was diverse and stable over time, with no evidence of CMV-responsive CD4 T cell expansion. We identified shared CDR3 TCR motifs among patients but no public CMV-specific TCRs. Temporal changes in clonality in response to transplantation and in the absence of detectable viral reactivation suggest changes in the repertoire immediately after transplantation followed by an expansion with stable clonal competition that may mediate protection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/fisiologia , Rejeição de Enxerto/imunologia , Transplante de Coração , Transplante de Rim , Adulto , Idoso , Antígenos Virais/imunologia , Proliferação de Células , Células Clonais , Feminino , Variação Genética , Humanos , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transplante Homólogo , Ativação Viral/imunologia
13.
Nat Microbiol ; 6(10): 1215-1216, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34545209

Assuntos
Células Clonais
14.
Parasitol Res ; 120(9): 3331-3333, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34345963

RESUMO

An alternative amoeba cloning technique is described. The amoebic cells were scraped from the agar and inoculated in a new agar medium separated in isolated squares, preventing a cell from invading the space of another cell, increasing the reliability of cloning, and providing an efficient quality control of technique.


Assuntos
Ágar , Amoeba , Meios de Cultura , Amoeba/crescimento & desenvolvimento , Células Clonais , Reprodutibilidade dos Testes
15.
J Clin Microbiol ; 59(10): e0094621, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34346714

RESUMO

Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time-consuming methods such as multilocus sequence typing or pulsed-field gel electrophoresis. Therefore, data on the prevalence of multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish epidemic strain A. ruhlandii (DES), are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. All the spectra of A. xylosoxidans (n = 1,571) and A. ruhlandii (n = 174) used to build the local database were analyzed by ClinProTools, MALDI Biotyper PCA, MALDI Biotyper dendrogram, and flexAnalysis software for biomarker peak detection. Two hundred two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Specific biomarker peaks were identified: absent peak at m/z 6,651 for AxST137 isolates and present peak at m/z 9,438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. The use of MALDI-TOF MS allowed us to identify isolates of A. xylosoxidans belonging to the AxST137 clone that spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help the implementation of segregation measures to avoid interpatient transmission of these resistant clones.


Assuntos
Achromobacter denitrificans , Achromobacter , Fibrose Cística , Epidemias , Achromobacter denitrificans/genética , Células Clonais , Fibrose Cística/complicações , Fibrose Cística/epidemiologia , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Cells ; 10(7)2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34359846

RESUMO

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line's most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.


Assuntos
Engenharia Celular/métodos , Proliferação de Células/genética , Edição de Genes/métodos , Transgenes , Sistemas CRISPR-Cas , Adesão Celular , Células Clonais , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transfecção
17.
Stem Cell Res ; 55: 102487, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34419748

RESUMO

Emery-Dreifuss muscular dystrophy type 1 (EDMD1) is a rare genetic disease caused by mutations in the EMD gene coding for a nuclear envelope protein emerin. We generated and characterized induced pluripotent stem cells (iPSCs) from two EDMD1 patients bearing a mutation c.del153C and from one healthy donor. That mutation leads to generation of premature STOP codon. Established iPSCs are very valuable tool for disease pathogenesis investigation and for the development of new therapeutic methods after differentiation to cardiac or muscle cells. Obtained iPSCs show the proper morphology, pluripotency markers expression, normal karyotype and potential to differentiate into three germ layers.


Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Emery-Dreifuss , Diferenciação Celular , Células Cultivadas , Células Clonais , Humanos , Distrofia Muscular de Emery-Dreifuss/genética , Mutação
18.
Ann Biol Clin (Paris) ; 79(4): 356-360, 2021 Aug 01.
Artigo em Francês | MEDLINE | ID: mdl-34427564

RESUMO

Flow cytometry is the gold standard for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clones. Several antibody panels have been used with varying sensitivities and specificities. The CD157 is one of the glycosylphosphatidylinositol-anchored molecules tested and widely used. The CD157 deficiency is rare. We report a case of an isolated CD157 deficiency discovered during the search for the PNH clone in a patient with a plastic anemia. The interpretation of the results in this case poses a problem of false positive. We discuss how to deal with these difficulties encountered by the biologist, detailing the various possible causes. This observation also underlines the importance of following international guidelines before making the diagnosis of the PNH clone which has significant implications.


Assuntos
Hemoglobinúria Paroxística , Células Clonais , Citometria de Fluxo , Hemoglobinúria Paroxística/diagnóstico , Humanos , Sensibilidade e Especificidade
19.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445118

RESUMO

The existence of CD4+ cytotoxic T cells (CTLs) at relatively high levels under different pathological conditions in vivo suggests their role in protective and/or pathogenic immune functions. CD4+ CTLs utilize the fundamental cytotoxic effector mechanisms also utilized by CD8+ CTLs and natural killer cells. During long-term cultivation, CD4+ T cells were also shown to acquire cytotoxic functions. In this study, CD4+ human T-cell clones derived from activated peripheral blood lymphocytes of healthy young adults were examined for the expression of cytotoxic machinery components. Cystatin F is a protein inhibitor of cysteine cathepsins, synthesized by CD8+ CTLs and natural killer cells. Cystatin F affects the cytotoxic efficacy of these cells by inhibiting the major progranzyme convertases cathepsins C and H as well as cathepsin L, which is involved in perforin activation. Here, we show that human CD4+ T-cell clones express the cysteine cathepsins that are involved in the activation of granzymes and perforin. CD4+ T-cell clones contained both the inactive, dimeric form as well as the active, monomeric form of cystatin F. As in CD8+ CTLs, cysteine cathepsins C and H were the major targets of cystatin F in CD4+ T-cell clones. Furthermore, CD4+ T-cell clones expressed the active forms of perforin and granzymes A and B. The levels of the cystatin F decreased with time in culture concomitantly with an increase in the activities of granzymes A and B. Therefore, our results suggest that cystatin F plays a role in regulating CD4+ T cell cytotoxicity. Since cystatin F can be secreted and taken up by bystander cells, our results suggest that CD4+ CTLs may also be involved in regulating immune responses through cystatin F secretion.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Cisteína/metabolismo , Inibidores de Proteases/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Catepsina C/metabolismo , Catepsina L/metabolismo , Linhagem Celular Tumoral , Células Clonais , Granzimas/metabolismo , Humanos , Células Jurkat , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/fisiologia , Linfócitos T Citotóxicos/metabolismo
20.
Cancer Immunol Res ; 9(6): 601, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34365414

RESUMO

Understanding the cellular regulation of tumor-specific CD8+ T-cell responses is critical to designing improved clinical strategies for cancer immunotherapy. In this issue, Aoki and colleagues deepen our knowledge of this topic by demonstrating that transient depletion of CD4+ T cells in patients with gastrointestinal cancer induces remodeling of the T-cell repertoire, including clonal replacement and expansion of CD8+ T-cell clones shared between the blood and tumor.See article by Aoki et al., p. 624.


Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Células Clonais , Humanos , Imunoterapia , Depleção Linfocítica
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