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1.
Braz. j. biol ; 82: e236471, 2022. tab, graf
Article in English | LILACS | ID: biblio-1249254

ABSTRACT

Abstract Date fruit is known to be the staple food in the Arab countries. It provides a lot of potential health benefits and can be the essential source of nutrients. The majority of Moroccan varieties are not characterized for their chemical, biochemical and quality properties. The aim of this work was to assess the chemical composition of 17 varieties of Moroccan date fruits (Phoenix dactylifera L.) and to determine their nutritive components. The analysis showed that the dates are rich in sugars (51.80-87.98%), they contain low concentration of proteins (1.09-2.80%) and lipids (0.16-0.39%). The predominant mineral is potassium (1055.26-1604.10 mg/100 g DW). Moreover, they contain high concentrations of malic acid (69.48-495.58 mg/100 g (DW)), oxalic acid (18.47-233.35 mg/100 g DW) and tartaric acid (115.70-484.168 mg/100 g DW). These results suggest that the date fruit are nutritious and can be an excellent source for human nutrition and health benefits.


Resumo A fruta da tâmara é conhecida por ser o alimento básico nos países árabes. Oferece muitos benefícios potenciais à saúde e pode ser a fonte essencial de nutrientes. A maioria das variedades marroquinas não se caracteriza por suas propriedades químicas, bioquímicas nem de qualidade. O objetivo deste trabalho foi avaliar a composição química de 17 variedades de frutos de tâmara marroquina (Phoenix dactylifera L.) e determinar seu valor nutritivo. A análise mostrou que as tâmaras são ricas em açúcares (51,80-87,98%) e contêm baixa concentração de proteínas (1,09-2,80%) e lipídios (0,16-0,39%). O mineral predominante é o potássio (1.055,26-1.604,10 mg/100 g DW). Além disso, contêm altas concentrações de ácido málico (69,48-495,58 mg/100 g DW), ácido oxálico (18,47-233,35 mg/100 g DW) e ácido tartárico (115,70-484,168 mg/100 g DW). Esses resultados sugerem que o fruto da tamareira é nutritivo e pode ser uma excelente fonte de nutrição humana e conferir benefícios à saúde.


Subject(s)
Humans , Phoeniceae , Clone Cells/chemistry , Fruit/chemistry , Minerals/analysis , Nutritive Value
2.
Article in English | WPRIM | ID: wpr-928604

ABSTRACT

OBJECTIVES@#To study the association between paroxysmal nocturnal hemoglobinuria (PNH) clone and immunosuppressive therapy (IST) in children with severe aplastic anemia (SAA).@*METHODS@#A retrospective analysis was performed on the medical data of 151 children with SAA who were admitted and received IST from January 2012 to May 2020. According to the status of PNH clone, these children were divided into a negative PNH clone group (n=135) and a positive PNH clone group (n=16). Propensity score matching was used to balance the confounding factors, and the impact of PNH clone on the therapeutic effect of IST was analyzed.@*RESULTS@#The children with positive PNH clone accounted for 10.6% (16/151), and the median granulocyte clone size was 1.8%. The children with positive PNH clone had an older age and a higher reticulocyte count at diagnosis (P<0.05). After propensity score matching, there were no significant differences in baseline features between the negative PNH clone and positive PNH clone groups (P>0.05). The positive PNH clone group had a significantly lower overall response rate than the negative PNH clone group at 6, 12, and 24 months after IST (P<0.05). The evolution of PNH clone was heterogeneous after IST, and the children with PNH clone showed an increase in the 3-year cumulative incidence rate of aplastic anemia-PNH syndrome (P<0.05).@*CONCLUSIONS@#SAA children with positive PNH clone at diagnosis tend to have poor response to IST and are more likely to develop aplastic anemia-PNH syndrome.


Subject(s)
Anemia, Aplastic/drug therapy , Child , Clone Cells , Hemoglobinuria, Paroxysmal/etiology , Humans , Immunosuppression Therapy , Retrospective Studies
3.
Chinese Journal of Biotechnology ; (12): 1218-1226, 2022.
Article in Chinese | WPRIM | ID: wpr-927776

ABSTRACT

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.


Subject(s)
Clone Cells , Mutagenesis, Site-Directed , Mutation , Plasmids/genetics , Polymerase Chain Reaction/methods
4.
Braz. J. Pharm. Sci. (Online) ; 58: e19692, 2022. graf
Article in English | LILACS | ID: biblio-1384014

ABSTRACT

Abstract The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.


Subject(s)
Tissue Plasminogen Activator , Process Optimization , Flow Cytometry/methods , Fluorescence , Cell Count/instrumentation , Clone Cells/classification , Plasminogen Activator Inhibitor 1/adverse effects , Green Fluorescent Proteins
5.
Electron. j. biotechnol ; 52: 67-75, July. 2021. tab, graf, ilus
Article in English | LILACS | ID: biblio-1283594

ABSTRACT

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.


Subject(s)
Cattle/metabolism , Adipocytes/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Fibroblasts/metabolism , Adipose Tissue/metabolism , Clone Cells , Cell Proliferation , Adipogenesis , Real-Time Polymerase Chain Reaction , Mitosis , Muscles
6.
São Paulo; s.n; s.n; 2021. 169 p. tab, ilus, graf.
Thesis in Portuguese | LILACS | ID: biblio-1382043

ABSTRACT

O aroma é um dos fatores mais importantes na determinação da qualidade e do caráter do vinho. Isso se deve à presença de compostos voláteis que estão associados às suas características organolépticas ou diferentes proporções entre estes compostos que podem ser influenciadas por fatores vitícolas (clima, solo, cultivar, manejo) e enológicos (maturação da uva, fermentação, tratamentos pósfermentativos). A região do sul de Minas Gerais vem se destacando na produção de espumantes de qualidade, e, nesse contexto, o presente trabalho teve como objetivo conhecer a influência do manejo da videira no desenvolvimento do aroma, da baga até o espumante, a fim de estabelecer associações com a qualidade do produto final. Os experimentos foram realizados com a cultivar Chardonnay em diferentes condições de manejo, em que foram avaliados clones, porta-enxertos, sistemas de condução e densidades de plantio. Foram analisados os compostos voláteis livres por HS-SPME/GC-MS das bagas, mostos, vinhos base e espumantes nas safras 2016, 2017 e 2018. O trabalho foi dividido em quatro partes para a apresentação dos resultados. A primeira consistiu em verificar a influência do material genético na composição volátil da cv. Chardonnay com os experimentos de clones e portaenxertos; a segunda parte avaliou a composição volátil do clone 809 até o espumante; a terceira, em analisar as vinificações dos diferentes sistemas de condução; e a quarta, em avaliar a evolução dos compostos voláteis da baga ao espumante e analisar os aromas que as densidades de plantio podem conferir ao espumante. As principais classes de compostos aromáticos identificados nas matrizes foram: C6-C9 aldeídos, álcoois superiores, aldeídos ramificados, benzenoides, monoterpenoides, norisoprenoides, sesquiterpenoides, cetonas e ácidos graxos. Os resultados mostraram que os clones e os porta-enxertos apresentaram perfis voláteis diferentes, indicando que a variabilidade entre os clones e que a enxertia têm influência no metabolismo da baga; o clone 809 apresenta maior abundância de compostos monoterpenoides, confirmando o seu caráter moscato, das uvas aos espumantes; os diferentes sistemas de condução e densidades de plantio alteram o metabolismo da14 baga, refletindo no perfil volátil dos espumantes nas safras estudadas. Dessa forma, os dados indicam que a composição volátil sofre influência do manejo da videira ao espumante


Aroma is one of the most important factors in determining the quality and character of wine. This is due to the presence of volatile compounds that are associated with their organoleptic characteristics or different proportions among these compounds that can be influenced by viticultural (climate, soil, cultivar, management) and oenological factors (grape maturation, fermentation, post fermentation treatments). The southern region of Minas Gerais has been standing out in the production of quality sparkling wines, and in this context, the purpose of the present work was to learn about the influence of grapevine management on the development of aroma, from berry to sparkling wine, in order to establish associations with the quality of the final product. The experiments were carried out with the Chardonnay cultivar under different management conditions, in which clones, rootstocks, trellising systems and planting densities were evaluated. The free volatile compounds by HS-SPME/GC-MS of the berries, musts, base and sparkling wines in the 2016, 2017 and 2018 harvests were analyzed. The work was divided into four parts in order to present the results. The first part consisted of verifying the influence of genetic material on the volatile composition of the cv. Chardonnay with the experiments on clones and rootstocks; the second part evaluated the volatile composition of clone 809 up to the sparkling wine; the third one part analyzed the vinification of the different trainig systems; and the fourth part evaluated the evolution of the volatile compounds from the berry to the sparkling wine and analyzed the aromas that the planting densities can confer to the sparkling wine. The main classes of aromatic compounds identified in the matrices were: C6-C9 aldehydes, higher alcohols, branched aldehydes, benzenoids, monoterpenoids, norisoprenoids, sesquiterpenoids, ketones and fatty acids. The results showed that the clones and the rootstocks have different volatile profiles, indicating that variability among clones and that grafting have great relevance to the berry secondary metabolism; the 809 clone presents a greater abundance of monoterpenoid compounds, confirming its muscat character, from grapes to sparkling wines; the different training systems and planting densities alter the berry´s metabolism, reflecting16 in the volatile profile of sparkling wines in the studied harvests. The data indicate that the volatile composition is influenced by the management of the berry to the sparkling wine


Subject(s)
Wine/adverse effects , Vitis/anatomy & histology , Foaming Agents , Agricultural Cultivation , Clone Cells/classification , Total Quality Management/methods , Fermentation , Fruit
7.
Rev. Bras. Cancerol. (Online) ; 67(3): e-091228, 2021.
Article in Portuguese | LILACS | ID: biblio-1292092

ABSTRACT

Introdução: O potencial de transformação maligna de células-tronco hematopoiéticas portadoras de mutações no gene glicosilfostatidilinositolclasse A (PIG-A) para leucemias agudas, embora raro, já é bem descrito na literatura. Objetivo: Neste estudo, porém, buscou-se evidenciar pela primeira vez na literatura o surgimento ou a manutenção de clones de hemoglobinúria paroxística noturna (HPN) em pacientes diagnosticados com leucemia aguda ou ainda após o início do tratamento quimioterápico. Método: A pesquisa de clones de HPN foi realizada por citometria de fluxo em blastos, hemácias, granulócitos ou monócitos de 47 amostras de sangue periférico e medula óssea de pacientes submetidos à investigação diagnóstica ou acompanhamento terapêutico, provenientes de dois hospitais oncológicos e públicos de Belém, no período de dezembro de 2017 a dezembro de 2018. Resultados: A presença de clones de HPN foi observada em 19/47 (40,4%) amostras de pacientes, em investigação diagnóstica ou acompanhamento terapêutico, que realizaram pelo menos um estudo de acompanhamento terapêutico e ainda tiveram o surgimento ou a manutenção do clone de HPN mesmo após iniciado o tratamento quimioterápico. Conclusão: Foi possível evidenciar, de forma primária, a presença de clones de HPN em pacientes diagnosticados com leucemia aguda tanto no período de investigação diagnóstica como durante o acompanhamento terapêutico, independentemente da ontogenia celular. Sem, porém, que se possa ainda avaliar a importância da presença desses clones de HPN para a evolução da doença primária, prognóstico ou necessidade de tratamento específico.


Introduction: The potential for malignant transformation of hematopoietic stem cells carrying mutations in theglycosylphosphatidylinositol class A (PIG-A) gene for acute leukemias, although rare, is already well described in the literature. Objective: In this study, however, it was attempted to show for the first time in the literature the emergence or maintenance of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients diagnosed with acute leukemia or even after the beginning of the chemotherapy treatment. Method: The search of PNH clones was performed by flow cytometry in blasts, erythrocytes, granulocytes or monocytes of 47 samples of peripheral blood and bone marrow from patients undergoing diagnostic investigation or therapeutic follow-up in two oncological and public hospitals in Belém, from December 2017 to December 2018. Results: The presence of PNH clones was observed in 19/47 (40.4%) patient samples, in diagnostic investigation or therapeutic follow-up, who participated of at least one therapeutic follow-up study and still experience the appearance or maintenance of the PNH clone even after the beginning of the chemotherapy treatment. Conclusion: Primarily, it was possible to demonstrate the presence of PNH clones in patients diagnosed with acute leukemia both during the diagnostic investigation period and therapeutic follow-up, regardless of cell ontogeny. However, the importance of the presence of these PNH clones for the evolution of the primary disease, prognosis or need for specific treatment was not evaluated yet.


Introducción: El potencial de transformación maligna de las células madre hematopoyéticas que portan mutaciones en el gen glicosofosfatidilinositol (GPI) clase A (PIGA) para las leucemias agudas, aunque raro, ya está bien descrito en la literatura. Objetivo: En este estudio, sin embargo, buscamos mostrar por primera vez en la literatura la aparición o mantenimiento de clones de HPN en pacientes diagnosticados de leucemia aguda o incluso después del inicio de la quimioterapia. Método: La investigación de clones de hemoglobinuria paroxística nocturna (HPN) se realizó mediante citometría de flujo en blastos, eritrocitos, granulocitos o monocitos de 47 muestras de sangre periférica y médula ósea de pacientes sometidos a investigación diagnóstica o seguimiento terapéutico de dos hospitales oncológicos y públicos de Belém, durante el período. de diciembre de 2017 a diciembre de 2018. Resultados: La presencia de clones HPN se observó en 19/47 (40,4%) muestras de pacientes, en investigación diagnóstica o seguimiento terapéutico, que realizaron al menos un estudio de seguimiento terapéutico y aún tenían la aparición o mantenimiento del clon HPN incluso después de iniciado el tratamiento de quimioterapia. Conclusión: Se pudo evidenciar, de forma primaria, la presencia de clones de HPN en pacientes diagnosticados de leucemia aguda tanto durante el período de investigación diagnóstica como durante el seguimiento terapéutico, independientemente de la ontogenia celular. Sin embargo, no podemos todavía evaluar la importancia de la presencia de estos clones de HPN para la evolución de la enfermedad primaria, el pronóstico o la necesidad de un tratamiento específico.


Subject(s)
Humans , Male , Female , Child, Preschool , Child , Adolescent , Adult , Middle Aged , Aged , Leukemia/diagnosis , Hemoglobinuria, Paroxysmal/blood , Bone Marrow/pathology , Leukemia/drug therapy , Clone Cells , Flow Cytometry , Hemoglobinuria, Paroxysmal/diagnosis
8.
Chinese Journal of Biotechnology ; (12): 178-186, 2021.
Article in Chinese | WPRIM | ID: wpr-878552

ABSTRACT

In order to establish an infectious clone for CDV-3, a commercial vaccine strain of canine distemper virus for mink, to provide reference for the studies of pathogenesis and novel vaccine development of CDV. Thirteen pairs of primers were used to amplify the full-length genome of CDV-3 strain. Five long fragments were obtained based on single restriction site analysis of the whole genome of CDV-3 by RT-PCR. Five fragments were successively inserted into the multiple clone sites in the modified eukaryotic vector of pcDNA3.2 by restriction enzymes and splicing. Meanwhile, the hammerhead ribozyme and hepatitis delta virus ribozyme sequences were added to the beginning of F1 fragment and the ending of F5 fragment, respectively. Then, the full-length cDNA recombinant plasmid of CDV-3 was obtained and named as pcDNA3.2-CDV-3. In addition, three helper plasmids, expressing the N protein, P protein and L protein of the CDV-3 strain respectively, were constructed. The 293T cells were transfected with the full-length cDNA recombinant plasmid and three helper plasmids by Lipofectamine™ 2000. At 3 days post transfection, the supernatant was added to the monolayer of Vero cells to observe the typical syncytium of CDV. Indirect immunofluorescence and artificial label identification of recombinant virus rCDV-3 were conducted after the occurrence of lesions. Finally, the growth characteristics of wtCDV-3 and rCDV-3 were compared after passaging of rCDV-3. The identification of the full-length cDNA recombinant plasmid and three helper plasmids by restriction enzyme digestion and sequencing were consistent with expected. The Vero cells infected with the recombinant rCDV-3 showed typical syncytic. The identification of indirect immunofluorescence and labeled marker, and observation under electron microscope proved that the rCDV-3 was indeed rescued from the recombinant plasmid of pcDNA3.2-CDV-3. In comparison of the virus titers of wtCDV-3, rCDV-3 replicated massively and rapidly and reached the maximize virus titer of 10⁷·⁶⁶⁷ TCID₅₀/mL within 36 h post infection (p.i.) in Vero cells, while wtCDV-3 grew gradually to 10⁶·⁶⁶⁷ TCID₅₀/mL at 72 h p.i. in Vero cells. This reverse genetic system of CDV-3 strain has been established successfully, to provide reference for the studies of pathogenesis and novel vaccine development of CDV.


Subject(s)
Animals , Chlorocebus aethiops , Clone Cells , DNA, Complementary , Distemper Virus, Canine/genetics , Plasmids/genetics , Vero Cells
9.
Pesqui. vet. bras ; 40(12): 1063-1072, Dec. 2020. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1155041

ABSTRACT

Somatic Cell Nuclear Transfer (SCNT-Cloning) is a promising technique in many areas and is based on genetically identical individuals. However, its efficiency is low. Studies suggest that the leading cause is inadequate epigenetic reprogramming. This study aimed to characterize the methylation pattern of the exon 10 regions of the IGF2 gene and the Imprinting Control Region (ICR) of the H19 gene in the placenta of cloned calves. For this study, female and male cloned calves presenting different phenotypes were used. Genomic DNA from these animals' placenta was isolated, then treated with sodium bisulfite and amplified to the ICR/H19 and IGF2 loci. PCR products were cloned into competent bacteria and finally sequenced. A significant difference was found between controls and clones with healthy phenotypes for the ICR/H19 region. In this region, controls showed a hemimethylated pattern, as predicted in the literature due to this region has an imprinted control, while clones were showed less methylated. For the IGF2, no significant differences were found between controls and clones. These results suggest that different genomic regions in the genome may be independently reprogrammed and that failures in reprogramming the DNA methylation patterns of imprinted genes may be one of the causes of the low efficiency of SCNT.(AU)


A Transferência Nuclear de Células Somáticas (TNCS-Clonagem) é uma técnica promissora em várias áreas, e se baseia na produção de indivíduos geneticamente idênticos. No entanto, sua eficiência é baixa. Estudos sugerem que a principal causa seja uma reprogramação epigenética inadequada. O objetivo desse trabalho é caracterizar o padrão de metilação da região éxon 10 do gene IGF2 e da Região Controladora de Imprinting (ICR) do gene H19 na placenta de bezerros clonados. Para a execução do trabalho foram selecionados clones bovinos fêmeas e machos, apresentando diferentes fenótipos. O DNA da placenta desses animais foi extraído, e em seguida foi tratado com bissulfito de sódio e amplificado para os loci ICR/H19 e IGF2. Os produtos da PCR foram clonados em bactérias competentes e, por fim, sequenciados. Foi encontrada uma diferença significativa entre os controles e os clones com fenótipos saudáveis para a região da ICR/H19. Nesta região, os controles tiveram um padrão hemimetilado, como previsto pela literatura, devido essa região ser imprinted. Enquanto os clones encontravam-se menos metilados. Para a região do éxon 10 do IGF2, não foi encontrada diferença significativa entre controles e clones. Estes resultados sugerem que as diferentes regiões do genoma podem se reprogramar independente umas das outras e que falhas na reprogramação do padrão de metilação do DNA de genes imprinted podem ser uma das causas da baixa eficiência da TNCS.(AU)


Subject(s)
Animals , Cattle , Placenta , Cattle/genetics , Clone Cells , Epigenomics , Insulin-Like Growth Factor II/analysis , DNA Methylation
10.
Biosci. j. (Online) ; 36(2): 619-627, 01-03-2020. ilus
Article in English | LILACS | ID: biblio-1146430

ABSTRACT

In this study E. coli recombinant clones that express the EC20 synthetic phytochelatin intracellularly were constructed. The increasement of Cd2+ biosorption capacity, and, also, the increasement of resistance to this toxic metal were analyzed. A gene that encodes the synthetic phytochelatin EC20 wassynthesized in vitro. The EC20 synthetic gene was amplified by PCR, inserted into the DNA cloning vectors pBluescript®KS+ and pGEM®-TEasy, and also into the expression vectors pTE [pET-28(a)® derivative] and pGEX-T4-2®. The obtained recombinant plasmids were employed for genetic transformation of E. coli: pBsKS-EC20 and pGEM-EC20, they were introduced into DH10B and DH5α strains, similarly to pTE-EC20 and pGEX-EC20 that were introduced into BL21 strain. The EC20 expression was confirmed by SDS-PAGE analysis. The recombinant clones' resistances to Cd2+ were determined by MIC analyses. The MIC for Cd2+ of DH10B/pBKS-EC20 and DH10B/pGEM-EC20 were 2.5 mM (EC20 induced), and 0.312 mM (EC20 repressed);respectively, 16 and 2 times higher than the control DH10B/pBsKS (0.156 mM). The MIC for Cd2+of BL21/pTE-EC20 was 10.0 mM (EC20 induced) and 2.5 mM (EC20 repressed), compared with the control BL21/pTE which was only 1.25 mM. Analysis of ICP-AES showed that BL21/pGEX-EC20, after growth on the condition of EC20 expression, absorbed 37.5% of Cd2+, and even when cultured into the non-induction condition of EC20 expression, it absorbed 11.5%.These results allow the conclusion thatrecombinant E. coli clonesexpressing the synthetic phytochelatin EC20 show increased capacity for Cd2+ biosorption and enhanced resistance to this toxic ion.


Foram construídos clones recombinantes de E. coli que expressam intracelularmente a fitoquelatina sintética EC20. Foi analisado o aumento na capacidade de biossorção de Cd2+ e o aumento da resistência a este metal tóxico.Foi sintetizado in vitro um gene codificante da fitoquelatina sintética EC20. O gene EC20 sintético foi amplificado por PCR, inserido nos vetores de clonagem pBluescript®KS+ e pGEM®-TEasy, e nos vetores de expressão pTE [derivado de pET-28(a)®] e pGEX-T4-2®. Os plasmídeos recombinantes foram empregados na transformação genética de E. coli: pBsKS-EC20 e pGEM-EC20 foram introduzidos nas linhagens DH10B e DH5α; e, pTE-EC20 e pGEX-EC20 na linhagem BL21-DE3. A expressão EC20 foi analisada por SDS-PAGE. As resistências a Cd2+ dos clones recombinantes foram determinadas por análises de MIC.A MIC para Cd2+ de DH10B/pBsKS-EC20 e de DH10B/pGEM-EC20 foi 2,5 mM (EC20induzido) e 0,312 mM (EC20 reprimido); respectivamente, 16 e 2 vezes superiores às do controle DH10B/pBsKS (0,156 mM). A MIC para Cd2+ de BL21/pTE-EC20 foi 10,0 mM (EC20 induzido) e 2,5 mM (EC20 reprimido), comparado a do controle BL21/pTE que foi apenas 1,25 mM. A análise de ICP-AES mostrou que BL21/pGEX-EC20, após crescimento na condição de expressão de EC20, absorveu 37,5% de Cd2+e, mesmo quando cultivado na condição de não-indução de expressão EC20, absorveu 11,5% de Cd2+. Estes resultados permitem a conclusão de que os clones recombinantes de E. coli que expressam a fitoquelatina sintética EC20 apresentam aumento da capacidade de biossorção de Cd2+ e de resistência a este íon tóxico.


Subject(s)
Cadmium , Escherichia coli , Phytochelatins , Biodegradation, Environmental , Clone Cells , Genetics
11.
J. appl. oral sci ; 28: e20200242, 2020. tab, graf
Article in English | LILACS, BBO | ID: biblio-1134786

ABSTRACT

Abstract Heterogeneous cell populations of osteo/cementoblastic (O/C) or fibroblastic phenotypes constitute the periodontal dental ligament (PDL). A better understanding of these PDL cell subpopulations is essential to propose regenerative approaches based on a sound biological rationale. Objective Our study aimed to clarify the differential transcriptome profile of PDL cells poised to differentiate into the O/C cell lineage. Methodology To characterize periodontal-derived cells with distinct differentiation capacities, single-cell-derived clones were isolated from adult human PDL progenitor cells and their potential to differentiate into osteo/cementoblastic (O/C) phenotype (C-O clones) or fibroblastic phenotype (C-F clones) was assessed in vitro. The transcriptome profile of the clonal cell lines in standard medium cultivation was evaluated using next-generation sequencing technology (RNA-seq). Over 230 differentially expressed genes (DEG) were identified, in which C-O clones showed a higher number of upregulated genes (193) and 42 downregulated genes. Results The upregulated genes were associated with the Cadherin and Wnt signaling pathways as well as annotated biological processes, including "anatomical structure development" and "cell adhesion." Both transcriptome and RT-qPCR showed up-regulation of WNT2, WNT16, and WIF1 in C-O clones. Conclusions This comprehensive transcriptomic assessment of human PDL progenitor cells revealed that expression of transcripts related to the biological process "anatomical structure development," Cadherin signaling, and Wnt signaling can identify PDL cells with a higher potential to commit to the O/C phenotype. A better understanding of these pathways and their function in O/C differentiation will help to improve protocols for periodontal regenerative therapies.


Subject(s)
Humans , Adult , Osteoblasts/cytology , Periodontal Ligament/surgery , Dental Cementum/cytology , Cadherins/metabolism , Cell Differentiation , Cells, Cultured , Clone Cells , Transcriptome
12.
Biol. Res ; 53: 13, 2020. tab, graf
Article in English | LILACS | ID: biblio-1100919

ABSTRACT

BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.


Subject(s)
Humans , Animals , Male , Middle Aged , Antigens, Tumor-Associated, Carbohydrate/genetics , Indians, South American/genetics , Gallbladder Neoplasms/genetics , Ascitic Fluid/metabolism , Tumor Cells, Cultured , Carcinogenicity Tests , Chile , DNA Fingerprinting , Tumor Suppressor Protein p53/genetics , Cisplatin/pharmacology , Mice, Inbred NOD , Clone Cells/drug effects , Clone Cells/metabolism , Sequence Analysis, RNA , Receptor, ErbB-2/genetics , Genes, erbB-2/genetics , Gene Expression Profiling , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Epithelial Cells/metabolism , Keratin-19/genetics , Keratin-7/genetics , Carcinogenesis/genetics , Gallbladder Neoplasms/metabolism , Antineoplastic Agents/pharmacology
13.
Article in English | WPRIM | ID: wpr-811275

ABSTRACT

Immunoglobulin G4 (IgG4)-related dacryoadenitis and sialoadenitis (IgG4-DS) are part of a multiorgan fibroinflammatory condition of unknown etiology termed IgG4-related disease (IgG4-RD), which has been recognized as a single diagnostic entity for less than 15 years. Histopathologic examination is critical for diagnosis of IgG4-RD. CD4+ T and B cells, including IgG4-expressing plasma cells, constitute the major inflammatory cell populations in IgG4-RD and are thought to cause organ damage and tissue fibrosis. Patients with IgG4-RD who have active, untreated disease exhibit significant increase of IgG4-secreting plasmablasts in the blood. Considerable insight into the immunologic mechanisms of IgG4-RD has been achieved in the last decade using novel molecular biology approaches, including next-generation and single-cell RNA sequencing. Exploring the interactions between CD4+ T cells and B lineage cells is critical for understanding the pathophysiology of IgG4-RD. Establishment of pathogenic T cell clones and identification of antigens specific to these clones constitutes the first steps in determining the pathogenesis of the disease. Herein, the clinical features and mechanistic insights regarding pathogenesis of IgG4-RD were reviewed.


Subject(s)
B-Lymphocytes , Clone Cells , Dacryocystitis , Diagnosis , Fibrosis , Humans , Immunoglobulins , Molecular Biology , Plasma Cells , Sequence Analysis, RNA , Sialadenitis , T-Lymphocytes
14.
São Paulo; s.n; 2020. 1-114 p. graf.
Thesis in Portuguese | LILACS, SES-SP, SESSP-TESESESSP, SES-SP | ID: biblio-1395595

ABSTRACT

O sorotipo O26:H11 associado às aEPEC e STEC tem sido frequentemente implicado com doenças entéricas em diversos países. Análises comparativas de cepas O26:H11 STEC e aEPEC de vários países indicam que as aEPEC O26:H11 representam clones que perderam os genes stx através da excisão fágica. No Brasil, o isolamento de aEPEC O26:H11 de casos de infecção humana é frequente, sendo este um dos mais importantes sorotipos de aEPEC em nosso meio. O objetivo deste estudo foi investigar e fazer uma análise comparativa sobre a ocorrência de vários genes de virulência, alguns deles altamente específicos para o patotipo STEC, em cepas O26:H11 STEC e aEPEC, além de avaliar a diversidade clonal através das técnicas de PFGE e sequenciamento MLST. Oito das 10 cepas STEC apresentaram o genótipo stx1a, enquanto duas cepas apresentaram stx2a. Este é o primeiro relato sobre a ocorrência de STEC O26:H11 albergando o genótipo stx2a no Brasil. Os genes plasmidiais ehxA, katP, espP e toxB foram encontrados em 8 (80%), 7 (70%), 8 (80%) e 8 (80%) das STEC. Todas as STEC abrigaram os genes efa, escN, nleB, nleE, sen, z2098, z2099, z2121, ureD e terE. Os genes espK e espM1 foram encontrados em igual frequência. Dentre as aEPEC, todas foram positivas para os genes efa, escN, nleB, nleE, sen e z2121. Os genes ehxA, espP, espM1, iha, katP, toxB, z2098, z2099, espK, espV, espN, ureD e terE estiveram presentes em 25 (66%), 25 (66%), 35 (92%), 31(82%), 16 (42%), 18 (47%), 26 (68%), 26 (68%), 31 (82%), 13 (34%), 19 (50%), 26 (68%) e 30 (79%) das cepas, respectivamente. O gene astA foi encontrado em apenas três (8%) aEPEC e nenhuma das cepas estudadas...(AU)


Subject(s)
Patient Isolation , Clone Cells , Shiga-Toxigenic Escherichia coli
15.
Arq. Inst. Biol ; 87: e0152019, 2020. tab
Article in English | LILACS, VETINDEX | ID: biblio-1130052

ABSTRACT

Root-knot nematode is one of the most important phytosanitary problems for Conilon coffee, as it reduces productivity and is difficult to handle. We aimed at studying the infectivity and damage caused by M. incognita race 1 in the "Jequitibá Incaper 8122" intermediate maturity coffee variety. The experiment was conducted in a greenhouse, in completely randomized design, with five replicates. The clones composing the variety "Jequitibá Incaper 8122" were inoculated with 2,000 eggs + second-stage juveniles of M. incognita race 1. Uninoculated plants were the control. Evaluations were performed 180 days after inoculation, considering the plant height (H), stem diameter (SD), number of leaves (NOL), leaf area (LA), number of plagiotropic branches (NPB), number of nodes (NN), chlorophyll content (CHLO), shoot dry matter (SDM), root fresh matter (RFM), final population (FNP), and reproduction factor (NRF). The nematode reduced NOL in clones 208 and 209, NRF in clones 201, 203, 207 and 208, NN in clones 203, 207, 208 and 209, CHLO in clones 201, 204, 206, 207 and 209, SDM in clones 201, 203, 204 and 205 and RFM in clones 205 and 207. M. incognita race 1 FNP and NRF were larger in clones 208, 201, 207 and 203. Clone 202 had FNP and NRF equal to zero, being immune to the nematode. Clone 206 presented the lowest NRF value among clones parasitized by M. incognita.(AU)


O nematoide-das-galhas é um dos mais importantes problemas fitossanitários para o cafeeiro conilon, por reduzir a produtividade e ser de difícil manejo. Objetivou-se estudar a infectividade e os danos causados por M. incognita raça 1 na variedade de café conilon de maturação intermediária "Jequitibá Incaper 8122". O experimento foi conduzido em casa de vegetação, em DIC, com cinco repetições. Os clones que compõem a variedade "Jequitibá Incaper 8122" foram inoculados com 2.000 ovos + juvenis de segundo estádio de M. incognita raça 1. Plantas não inoculadas constituíram a testemunha. As avaliações foram realizadas 180 dias após a inoculação, sendo avaliados: altura da planta (ALT), diâmetro do caule (DCA), número de folhas (NFO), área foliar (AFO), número de ramos plagiotrópicos (NRP), número de nós (NN), teor de clorofila (CLO), massa seca da parte aérea (MSA), matéria fresca da raiz (MFR), população final (PFN) e fator de reprodução (FRE). O nematoide reduziu o NFO nos clones 208 e 209, NRP nos clones 201, 203, 207 e 208, NN nos clones 203, 207, 208 e 209, CLO nos clones 201, 204, 206, 207 e 209, MSA nos clones 201, 203, 204 e 205 e MFR nos clones 205 e 207. PFN e FRE de M. incognita raça 1 foram maiores nos clones 208, 201, 207 e 203; o clone 202 teve a PFN e a FRE igual a zero, apresentando-se imune ao nematoide. O clone 206 apresentou o menor valor de FRE entre os clones parasitados por M. incognita.(AU)


Subject(s)
Coffee Industry , Coffea , Nematoda , Tylenchoidea , Pest Control , Clone Cells , Microscopy, Electron, Scanning Transmission , Coffee , Agricultural Pests
16.
Braz. j. med. biol. res ; 53(12): e9317, 2020. graf
Article in English | LILACS, ColecionaSUS | ID: biblio-1132508

ABSTRACT

LINC00355 has been reported aberrantly over-expressed and associated with poor prognosis in various types of cancer. However, reports regarding the effect of LINC00355 on lung squamous cell carcinoma (SCC) are rare. This study aimed to explore the function of LINC00355 in the development and progression of lung SCC and reveal the underlying mechanism. The expression and subcellular location of LINC00355 were determined by qRT-PCR and RNA-FISH, respectively. The lung SCC cell growth was analyzed by CCK-8 assay, transwell invasion, wound healing, colony formation, and flow cytometry assays. Reactive oxygen species level was evaluated by DCFH-DA probes. Bioinformatics online websites, luciferase reporter assay, RNA binding protein immunoprecipitation (RIP), and RNA pull-down assays were utilized to investigate the interaction among LINC00355, miR-466, and Ly-1 antibody reactive clone (LYAR). The results showed that LINC00355 was upregulated in lung SCC and was positively associated with poor overall survival in lung SCC patients. LINC00355 was mainly located in the cytoplasm of SCC cells. Additionally, LINC0035 functioned as a competing endogenous RNA (ceRNA) to target miR-466, and LYAR was identified as a direct target of miR-466. LINC00355 expression negatively correlated with miR-466 level, and positively correlated with LYAR level. Mechanistically, knockdown of LINC00355 inhibited cell proliferation, migration and invasion, promoted cell apoptosis in vitro, and suppressed tumor growth in vivo through targeting miR-466, and thus down-regulated LYAR expression. These findings provide a new sight for understanding the molecular mechanism of lung SCC and indicate that LINC00355 may serve as a potential biomarker for the diagnosis and treatment of lung SCC.


Subject(s)
Humans , Carcinoma, Squamous Cell/genetics , RNA, Long Noncoding/genetics , Lung Neoplasms/genetics , Nuclear Proteins , Gene Expression Regulation, Neoplastic , Clone Cells , MicroRNAs , Cell Line, Tumor , DNA-Binding Proteins , Lung
17.
Biomédica (Bogotá) ; 39(2): 291-299, ene.-jun. 2019. tab, graf
Article in Spanish | LILACS | ID: biblio-1011441

ABSTRACT

Resumen Introducción. Se ha demostrado que el caseinato de sodio y sus componentes (caseínas α, β y κ) inhiben la proliferación de la línea celular hematopoyética de ratón 32D clone 3 (32Dcl3) e inducen su diferenciación hacia macrófagos. Se sabe que la caseína α induce la producción de IL-1β y que esta última citocina inhibe la proliferación celular mediante la producción del factor de necrosis tumoral alfa (TNF-α), pero se desconoce si el caseinato de sodio y las caseínas inducen la producción de TNF y si este es el responsable de la inhibición de la proliferación. Objetivo. Evaluar si el caseinato de sodio y las caseínas α, β y κ inhiben la proliferación de la línea celular 32Dcl3 mediante la producción de TNF-α. Materiales y métodos. Se usaron diferentes concentraciones de caseinato de sodio y de las caseínas α, β y κ en las células 32Dcl3. Posteriormente, se evaluaron la viabilidad celular mediante una prueba con el MTT [3-(4,5-dimetiltiazol-2-ilo)-2,5-difeniltetrazol], la inducción de apoptosis con la citometría de flujo y la síntesis del TNF-α con el ELISA. Además, se hicieron pruebas de neutralización con anti-TNF-α en células 32Dcl3 tratadas con caseinato de sodio y caseína α, y se evaluó la proliferación celular. Resultados. Se encontró que el caseinato de sodio y las caseínas α, β y κ reducían la proliferación de la línea celular 32Dcl3 sin afectar la viabilidad, y que solo el caseinato y la caseína α inducían la apoptosis y la liberación al medio de TNF-α. La proliferación de células 32Dcl3 tratadas con caseinato y caseína α se restableció al usar anticuerpos anti-TNF-α. Conclusión. El TNF-α fue el principal responsable de la inhibición de la proliferación en las células 32Dcl3 tratadas con caseinato de sodio o caseína α.


Abstract Introduction: Sodium caseinate (CS) and its components (alpha-casein, beta-casein, and kappa-casein) have been shown to inhibit the proliferation of the mouse hematopoietic 32D clone 3 (32Dcl3) cell line and induce its differentiation into macrophages. It is well-known that alpha-casein induces IL-1β production and that this cytokine inhibits the proliferation via the production of tumor necrosis factor alpha (TNF-alpha), but it is not known if CS and the caseins inhibit the proliferation via TNF-alpha production. Objective: To evaluate if CS and alpha-casein, beta-casein and kappa-casein inhibit the proliferation on 32Dcl3 cell line via TNF-alpha. Materials and methods: We used different concentrations of CS, alpha-casein, beta-casein and kappa-casein in 32Dcl3 cells to evaluate cell proliferation. We assessed cell viability by MTT, induction to apoptosis by flow cytometry, and TNF-alpha synthesis by ELISA. Additionally, we performed anti-TNF-alpha neutralization assays on 32Dcl3 cells treated with CS and alpha-casein and we evaluated proliferation. Results: The results showed that CS, alpha-casein, beta-casein, and kappa-casein reduced proliferation of the 32Dcl3 cell line without affecting the viability and that only CS and alpha-casein induced apoptosis and the release of TNF-alpha. The 32Dcl3 cells treated with CS and alpha-casein reestablished their proliferation by using anti-TNF-alpha antibodies. Conclusion: TNF-alpha was the main responsible for the inhibition of proliferation in 32Dcl3 cells treated with CS or alpha-casein.


Subject(s)
Animals , Mice , Caseins/pharmacology , Tumor Necrosis Factor-alpha/physiology , Myeloid Cells/drug effects , Myelopoiesis/drug effects , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis , Clone Cells , Apoptosis/drug effects , Myeloid Cells/cytology , Macrophages/cytology
18.
Article in English | WPRIM | ID: wpr-758907

ABSTRACT

This study examined the effects of a caffeine treatment to improve nuclear reprogramming in porcine cloned embryos. Embryonic development and the expression of genes related to pluripotency (POU5F1, SOX2, NANOG, and CDX2) were compared after caffeine supplementation during manipulation at different concentrations (0, 1.25, 2.5, and 5.0 mM) and after varying the delayed activation time (control, 1, 2, and 4 h) after fusion. Caffeine added to media during manipulation produced a higher rate of development to blastocysts in the 1.25 mM group than in the other concentration groups (22.8% vs. 16.1%, 16.2%, and 19.2%; p < 0.05). When caffeine was added during the 4 h delayed activation, the 1.25 mM caffeine concentration produced a significantly higher rate of development than those in the other 4 h-activation-delayed caffeine concentration groups (22.4% vs. 9.4%, 14.0%, and 11.1%; p < 0.05). On the other hand, no significant improvement over that in the control group was observed when caffeine was supplemented during both the manipulation period and delayed activation period (16.0% vs. 15.2%), respectively. The levels of POU5F1, SOX2, and NANOG expression in blastocysts were significantly higher in the delayed activation caffeine group (4 h, 1.25 mM) than in the control group (1 h, 0 mM; p < 0.05). In conclusion, a caffeine treatment at 1.25 mM during delayed activation for 4 h can improve the preimplantation development of porcine somatic cell nuclear transfer embryos by activating nuclear reprogramming.


Subject(s)
Blastocyst , Caffeine , Cellular Reprogramming , Clone Cells , Embryonic Development , Embryonic Structures , Female , Hand , Pregnancy
19.
Article in English | WPRIM | ID: wpr-758905

ABSTRACT

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.


Subject(s)
Actinobacillus pleuropneumoniae , Actinobacillus , Animals , Antibodies , Blotting, Western , Clone Cells , Enzyme-Linked Immunosorbent Assay , Guinea Pigs , Methods , Recombinant Proteins , Vaccines , Vaccines, Subunit
20.
Article in English | WPRIM | ID: wpr-758879

ABSTRACT

Sequence type (ST) 33 of Shiga toxin-producing Escherichia coli (STEC) strain O91:H14 has been proposed as a potential domestic clone of STEC in Korea because of its high prevalence among human patients with mild diarrhea or asymptomatic carriers. Herein, the clonal diversity of 17 STEC O91:H14 isolates of ST33 during 2003 to 2014 was analyzed by pulsed-field gel electrophoresis, including 14 isolates from human patients and 3 from retail meats. Their virulence characteristics, acid resistance, and antimicrobial susceptibility were also determined. Our results showed that all isolates were clustered mainly into three different pulsotypes and were likely low pathogenic without antimicrobial resistance.


Subject(s)
Clone Cells , Diarrhea , Electrophoresis, Gel, Pulsed-Field , Escherichia coli , Humans , Korea , Meat , Molecular Epidemiology , Prevalence , Shiga Toxin , Shiga-Toxigenic Escherichia coli , Virulence
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