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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.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
Braz. j. med. biol. res ; 53(12): e9317, 2020. graf
Article in English | ColecionaSUS, 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
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.
Article in Korean | WPRIM | ID: wpr-760490

ABSTRACT

BACKGROUND: Flow cytometry analysis of paroxysmal nocturnal hemoglobinuria (PNH) is significantly affected by the methodology used. The lack of data on the effect of age and refrigeration on PNH clone stability motivated us to study these aspects using flow cytometry. METHODS: Peripheral blood was collected from six patients, of which two presented with PNH. All samples were tested immediately and stored at room temperature (RT, 20–25℃) and at 4℃ for re-analysis at 24, 48, 72 hr and 7 days. Anti-CD59-fluorescein isothiocyanate (Beckman Coulter, USA) and anti-CD235a-phycoerythrin (PE; Beckman Coulter) were used to stain red blood cells (RBCs). Fluorescein-labeled proaerolysin (Cedarlane, Canada), anti-CD15-PE (Beckman Coulter), anti-CD24-PE-cyanin 5 (Beckman Coulter), and anti-CD45-PE-cyanin 7 (Beckman Coulter) were used to stain granulocytes. Flow cytometry was performed using a FC500 flow cytometer (Beckman Coulter). The effects of time and temperature were analyzed using generalized estimating equations. RESULTS: No significant differences in the gated percentage of RBCs and PNH clone size of RBCs were observed between the RT and 4℃ groups up to 7 days of testing. The percentage of gated neutrophils decreased with specimen age (P<0.001) and a better correlation with baseline was obtained at 4℃ than at RT (P=0.014). Neutrophil PNH clones were stable until 48 hr and 72 hr at RT and 4℃, respectively, and could not be analyzed at 7 days. CONCLUSIONS: RBC analysis was successfully performed up to 7 days. For neutrophils, testing within 48 hr is recommended, because the number of gated cells decreases significantly with age.


Subject(s)
Clone Cells , Erythrocytes , Flow Cytometry , Granulocytes , Hemoglobinuria, Paroxysmal , Humans , Neutrophils , Refrigeration
13.
Article in English | WPRIM | ID: wpr-759593

ABSTRACT

BACKGROUND: The recent expansion of knowledge about various ABO alleles has led to the need for a comprehensive measure to cover the numerous polymorphisms dispersed in the ABO gene. A few studies have examined the diversity of the O allele compared to A or B subgroup alleles, resulting in antigenic changes. This study investigated the relationship between the serologic and molecular genetic characteristics of the O alleles in the Korean population. METHODS: One hundred and five samples from healthy blood group O subjects were selected randomly. The isoagglutinin titer was measured using a tube agglutination and gel microcolumn assay. The ABO alleles were analyzed by sequencing exons 6 and 7 of the ABO gene. When the origin of a heterozygous nucleotide sequence was ambiguous, it was separated into a single allele using mono-allele amplification or cloning. RESULTS: The median IgM isoagglutinin titer was eight. In contrast, the median IgG anti-A and anti-B isoagglutinin titers were 64 and 32, respectively. The IgG isoagglutinin titer showed a significant increase with age (P<0.0001). Six O alleles were observed in 105 blood group O populations by sequencing. The O01 and O02 alleles were common (0.57, 0.36). Three rare O alleles (O04, O05, and O06) and one novel non-deletional O allele were found. CONCLUSION: The distribution of isoagglutinin titers of blood group O and the genetic frequency of O alleles in this study would form the basis of the development and interpretation of ABO genotyping and serologic workup in the Korean population.


Subject(s)
Agglutination , Alleles , Base Sequence , Clone Cells , Cloning, Organism , Exons , Immunoglobulin G , Immunoglobulin M , Molecular Biology , Sequence Analysis
14.
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
15.
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
16.
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
19.
Article in English | WPRIM | ID: wpr-763368

ABSTRACT

PURPOSE: Canine influenza virus (CIV), H3N2, carries potentiality for zoonotic transmission and genetic assortment which raises a concern on possible epidemics, and human threats in future. To manage possible threats, the development of rapid and effective methods of CIV vaccine production is required. The plant provides economical, safe, and robust production platform. We investigated whether hemagglutinin (HA) antigen from Korea-originated CIV could be produced in Nicotiana benthamiana and lettuce, Lactuca sativa by a DNA viral vector system. MATERIALS AND METHODS: We used DNA sequences of the HA gene from Korean CIV strain influenza A/canine/Korea/S3001/2015 (H3N2) for cloning into a geminiviral expression vectors to express recombinant HA (rHA) antigen in the plant. Agrobacterium-mediated infiltration was performed to introduce HA-carrying vector into host plants cells. Laboratory-grown N. benthamiana, and grocery-purchased or hydroponically-grown lettuce plant leaves were used as host plants. RESULTS: CIV rHA antigen was successfully expressed in host plant species both N. benthamiana and L. sativa by geminiviral vector. Both complex-glycosylated and basal-glycosylated form of rHA were produced in lettuce, depending on presence of endoplasmic reticulum (ER) retention signal. In terms of rHA expression level, canine HA (H3N2) showed preference to the native signal peptide than ER retention signal peptide in the tested geminiviral vector system. CONCLUSION: Grocery-purchased lettuce leaves could serve as an instant host system for the transient expression of influenza antigen at the time of emergency. The geminiviral vector was able to induce expression of complex-glycosylated and basal-glycosylated rHA in lettuce and tobacco.


Subject(s)
Base Sequence , Clone Cells , Cloning, Organism , DNA , Emergencies , Endoplasmic Reticulum , Hemagglutinins , Humans , Influenza, Human , Lettuce , Orthomyxoviridae , Plant Leaves , Plants , Protein Sorting Signals , Tobacco
20.
Article in English | WPRIM | ID: wpr-763192

ABSTRACT

PURPOSE: This study was carried out to identify a peptide that selectively binds to kidney injury molecule-1 (KIM-1) by screening a phage-displayed peptide library and to use the peptide for the detection of KIM-1overexpressing tumors in vivo. MATERIALS AND METHODS: Biopanning of a phage-displayed peptide library was performed on KIM-1–coated plates. The binding of phage clones, peptides, and a peptide multimer to the KIM-1 protein and KIM-1–overexpressing and KIM-1–low expressing cells was examined by enzyme-linked immunosorbent assay, fluorometry, and flow cytometry. A biotin-peptide multimer was generated using NeutrAvidin. In vivo homing of the peptide to KIM-1–overexpressing and KIM1–low expressing tumors in mice was examined by whole-body fluorescence imaging. RESULTS: A phage clone displaying the CNWMINKEC peptide showed higher binding affinity to KIM-1 and KIM-1–overexpressing 769-P renal tumor cells compared to other phage clones selected after biopanning. The CNWMINKEC peptide and a NeutrAvidin/biotin-CNWMINKEC multimer selectively bound to KIM-1 over albumin and to KIM-1–overexpressing 769-P cells and A549 lung tumor cells compared to KIM-1–low expressing HEK293 normal cells. Co-localization and competition assays using an anti–KIM-1 antibody demonstrated that the binding of the CNWMINKEC peptide to 769-P cells was specifically mediated by KIM-1. The CNWMINKEC peptide was not cytotoxic to cells and was stable for up to 24 hours in the presence of serum. Whole-body fluorescence imaging demonstrated selective homing of the CNWM-INKEC peptide to KIM-1–overexpressing A498 renal tumor compared to KIM1–low expressing HepG2 liver tumor in mice. CONCLUSION: The CNWMINKEC peptide is a promising probe for in vivo imaging and detection of KIM-1‒overexpressing tumors.


Subject(s)
Animals , Bacteriophages , Clone Cells , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Fluorometry , Kidney Neoplasms , Kidney , Liver , Lung , Mass Screening , Mice , Optical Imaging , Peptide Library , Peptides
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