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Electron. j. biotechnol ; 52: 35-44, July. 2021. tab, ilus
Article in English | LILACS | ID: biblio-1283494


BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

Polysaccharide-Lyases/metabolism , Oxygen Transfer , Azotobacter vinelandii/metabolism , Oxygen/metabolism , Gene Expression , Polymerase Chain Reaction , Azotobacter vinelandii/genetics , Alginates/metabolism , Fermentation , Molecular Weight
Electron. j. biotechnol ; 52: 21-29, July. 2021. ilus, tab, graf
Article in English | LILACS | ID: biblio-1283484


BACKGROUND: Super-paramagnetic iron oxide nanoparticles (SPION) contain a chemotherapeutic drug and are regarded as a promising technique for improving targeted delivery into cancer cells. RESULTS: In this study, the fabrication of 5-fluorouracil (5-FU) was investigated with loaded Dextran (DEXSPION) using the co-precipitation technique and conjugated by folate (FA). These nanoparticles (NPs) were employed as carriers and anticancer compounds against liver cancer cells in vitro. Structural, magnetic, morphological characterization, size, and drug loading activities of the obtained FA-DEX-5-FUSPION NPs were checked using FTIR, VSM, FESEM, TEM, DLS, and zeta potential techniques. The cellular toxicity effect of FA-DEX-5-FU-SPION NPs was evaluated using the MTT test on liver cancer (SNU-423) and healthy cells (LO2). Furthermore, the apoptosis measurement and the expression levels of NF-1, Her-2/neu, c-Raf-1, and Wnt-1 genes were evaluated post-treatment using flow cytometry and RT-PCR, respectively. The obtained NPs were spherical with a suitable dispersity without noticeable aggregation. The size of the NPs, polydispersity, and zeta were 74 ± 13 nm, 0.080 and 45 mV, respectively. The results of the encapsulation efficiency of the nano-compound showed highly colloidal stability and proper drug maintenance. The results indicated that FA-DEX-5-FU-SPION demonstrated a sustained release profile of 5-FU in both phosphate and citrate buffer solutions separately, with higher cytotoxicity against SNU-423 cells than against other cells types. These findings suggest that FA-DEX-SPION NPs exert synergistic effects for targeting intracellular delivery of 5-FU, apoptosis induction, and gene expression stimulation. CONCLUSIONS: The findings proved that FA-DEX-5-FU-SPION presented remarkable antitumor properties; no adverse subsequences were revealed against normal cells.

Humans , Carcinoma, Hepatocellular/drug therapy , Fluorouracil/administration & dosage , Liver Neoplasms/drug therapy , Polymers , Gene Expression/drug effects , Drug Delivery Systems , Apoptosis/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Delayed-Action Preparations , Nanoparticles/administration & dosage , Magnetite Nanoparticles , Flow Cytometry
Rev. bras. med. esporte ; 27(spe2): 50-53, Apr.-June 2021. graf
Article in English | LILACS | ID: biblio-1280084


ABSTRACT Extraction of effective components from Pueraria lobata has important value for skeletal muscle quality and gene expression. The improvement effect of traditional high-intensity intermittent training on skeletal muscle has not been obvious, and it is difficult to guarantee the properties of some volatiles. Based on this, this paper analyzes the effect of high-intensity intermittent training on skeletal muscle quality and gene expression in Pueraria lobata. Based on a brief summary of extraction of Pueraria lobata, status of research on the pharmaceutical components of Pueraria lobata was summarized. Different specimens of Pueraria lobata were selected as research objects, and the process of high-intensity intermittent training was designed. High-intensity intermittent training, solvent extraction and water solvent extraction were combined together to design the fixed-bed continuous extraction scheme. According to the influence of Pueraria lobata on skeletal muscle quality, the influence of intermittent training on skeletal muscle quality was analyzed. The extraction results showed that Pueraria lobata combined with high-intensity intermittent training can effectively improve the content of skeletal muscle and ensure the effective expression of skeletal muscle gene.

RESUMO A extração de componentes eficazes da Pueraria lobata tem importante valor para a qualidade músculoesquelética e para a expressão genética. O efeito da melhoria do tradicional treinamento intervalado de alta intensidade na estrutura músculoesquelética não tem sido óbvio, e é difícil garantir as propriedades de alguns voláteis. Com base nisso, este estudo analisa o efeito do treinamento intervalado de alta intensidade na qualidade músculoesquelética e na expressão genética na Pueraria lobata. Com base num breve resumo da extração da Pueraria lobata, resumiu-se o andamento das pesquisas sobre os componentes farmacêuticos da Pueraria lobata. Diferentes amostras de Pueraria lobata foram selecionadas como objeto de pesquisa, e formulou-se o processo do treinamento intervalado de alta intensidade. O treinamento intervalado de alta intensidade, a extração de solventes e a extração de solventes à base de água foram combinadas para conceber o sistema de extração contínua de leito fixo. De acordo com a influência da Pueraria lobata na qualidade músculoesquelética, analisou-se a influência do treino intervalado na qualidade músculoesquelética. Os resultados da extração mostraram que a Pueraria lobata, combinada com treino intervalado de alta intensidade, pode melhorar, de maneira eficaz, o teor músculoesquelético e garantir a expressão eficaz da expressão genética do músculoesquelético.

RESUMEN La extracción de componentes eficaces de la Pueraria lobata tiene un importante valor para la calidad músculoesquelética y para la expresión genética. El efecto de la mejora del tradicional entrenamiento intercalado de alta intensidad en la estructura músculoesquelética no ha sido obvio, y es difícil garantizar las propriedades de algunos volátiles. Basándose en eso, este estudio analiza el efecto del entrenamiento intercalado de alta intensidad en la calidad músculoesquelética y en la expresión genética en la Pueraria lobata. Basándose en un breve resumen de la extracción de la Pueraria lobata, se resumió el andamiento de las investigaciones sobre los componentes farmacéuticos de la Pueraria lobata. Diferentes muestras de Pueraria lobata fueron seleccionadas como objeto de investigación, y se formuló el proceso del entrenamiento intercalado de alta intensidad. El entrenamiento intercalado de alta intensidad, la extracción de solventes y la extracción de solventes a base de agua fueron combinadas para concebir el sistema de extracción continua de lecho fijo. De acuerdo con la influencia de la Pueraria lobata en la calidad músculoesquelética, se analizó la influencia del entrenamiento intercalado en la calidad músculoesquelética. Los resultados de la extracción mostraron que la Pueraria lobata, combinada con entrenamiento intercalado de alta intensidad, puede mejorar, de manera eficaz, el tenor músculoesquelético y garantizar la expresión eficaz de la expresión genética del músculoesquelético.

Humans , Plant Extracts/administration & dosage , Gene Expression , Muscle, Skeletal/drug effects , Pueraria/chemistry , High-Intensity Interval Training/methods
Electron. j. biotechnol ; 51: 50-57, May. 2021. ilus, graf
Article in English | LILACS | ID: biblio-1343384


BACKGROUND: Molecular brain therapies require the development of molecular switches to control gene expression in a limited and regulated manner in time and space. Light-switchable gene systems allow precise control of gene expression with an enhanced spatio-temporal resolution compared to chemical inducers. In this work, we adapted the existing light-switchable Light-On system into a lentiviral platform, which consists of two modules: (i) one for the expression of the blue light-switchable transactivator GAVPO and (ii) a second module containing an inducible-UAS promoter (UAS) modulated by a light-activated GAVPO. RESULTS: In the HEK293-T cell line transfected with this lentiviral plasmids system, the expression of the reporter mCherry increased between 4 to 5 fold after light induction. A time expression analysis after light induction during 24 h revealed that mRNA levels continuously increased up to 9 h, while protein levels increased throughout the experiment. Finally, transduction of cultured rat hippocampal neurons with this dual Light-On lentiviral system showed that CDNF, a potential therapeutic trophic factor, was induced only in cells exposed to blue light. CONCLUSIONS: In conclusion, the optimized lentiviral platform of the Light-On system provides an efficient way to control gene expression in neurons, suggesting that this platform could potentially be used in biomedical and neuroscience research, and eventually in brain therapies for neurodegenerative diseases.

Gene Expression Regulation , Optogenetics/methods , Light , Neurons/metabolism , Immunoblotting , Gene Expression , Fluorescent Antibody Technique , Lentivirus
Rev. Hosp. Ital. B. Aires (2004) ; 41(1): 37-42, mar. 2021. ilus, tab
Article in Spanish | LILACS | ID: biblio-1178964


El término CRISPR, por su acrónimo en inglés refiere a Clustered Regularly Interspaced Short Palindromic Repeats, es decir, repeticiones palindrómicas cortas, agrupadas y regularmente esparcidas, por sus características en el genoma, pertenece naturalmente al sistema de defensa de bacterias y arqueas. Este ha sido adaptado biotecnológicamente para la edición del ADN de células eucariotas, incluso de células humanas. El sistema CRISPR-Cas para editar genes consta, en forma generalizada, de dos componentes: una proteína nucleasa (Cas) y un ARN guía (sgRNA). La simplicidad del complejo lo hace una herramienta molecular reprogramable capaz de ser dirigida y de editar cualquier sitio en un genoma conocido. Su principal foco son las terapias para enfermedades hereditarias monogénicas y para el cáncer. Sin embargo, además de editor de genes, la tecnología CRISPR se utiliza para edición epigenética, regulación de la expresión génica y método de diagnóstico molecular. Este artículo tiene por objetivo presentar una revisión de las aplicaciones de la herramienta molecular CRISPR-Cas, particularmente en el campo biomédico, posibles tratamientos y diagnósticos, y los avances en investigación clínica, utilizando terapia génica con CRISPR/Cas más relevantes hasta la fecha. (AU)

CRISPR are Clustered Regularly Interspaced Short Palindromic Repeats, which naturally belong to the defense system of bacteria and archaea. It has been biotechnologically adapted for editing the DNA of eukaryotic cells, including human cells. The CRISPR-Cas system for editing genes generally consists of two components, a nuclease protein (Cas) and a guide RNA (sgRNA). The simplicity of the complex makes it a reprogrammable molecular tool capable of being targeted and editing any site in a known genome. Its main focus is therapies for monogenic inherited diseases and cancer. However, in addition to gene editor, CRISPR technology is used for epigenetic editing, regulation of gene expression, and molecular diagnostic methods. This article aims to present a review of the applications of the CRISPR-Cas molecular tool, particularly in the biomedical field, possible treatments and diagnoses, and the advances in clinical research, using the most relevant CRISPR-Cas gene therapy to date. (AU)

Humans , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , CRISPR-Cas Systems/genetics , Biotechnology , Genetic Therapy/methods , Gene Expression , Genome, Human/genetics , Gene Expression Regulation , Epigenomics/trends , CRISPR-Associated Proteins/genetics , CRISPR-Associated Proteins/therapeutic use , Genetic Diseases, Inborn/therapy , Neoplasms/therapy
Electron. j. biotechnol ; 50: 59-67, Mar. 2021. ilus, graf, tab
Article in English | LILACS | ID: biblio-1292412


BACKGROUND: Cross talk of tumor­immune cells at the gene expression level has been an area of intense research. However, it is largely unknown at the alternative splicing level which has been found to play important roles in the tumor­immune microenvironment. RESULTS: Here, we re-exploited one transcriptomic dataset to gain insight into tumor­immune interactions from the point of AS level. Our results showed that the AS profiles of triple-negative breast cancer cells co-cultured with activated T cells were significantly changed but not Estrogen receptor positive cells. We further suggested that the alteration in AS profiles in triple-negative breast cancer cells was largely caused by activated T cells rather than paracrine factors from activated T cells. Biological pathway analyses showed that translation initiation and tRNA aminoacylation pathways were most disturbed with T cell treatment. We also established an approach largely based on the AS factor­AS events associations and identified LSM7, an alternative splicing factor, may be responsible for the major altered events. CONCLUSIONS: Our study reveals the notable differences of response to T cells among breast cancer types which may facilitate the development or improvement of tumor immunotherapy.

T-Lymphocytes , Triple Negative Breast Neoplasms , Peptide Chain Initiation, Translational , Gene Expression , Alternative Splicing , Cell Culture Techniques , Receptor Cross-Talk , Transfer RNA Aminoacylation , Transcriptome , Immunotherapy
Electron. j. biotechnol ; 50: 68-76, Mar. 2021. ilus, tab, graf
Article in English | LILACS | ID: biblio-1292417


BACKGROUND: Jasmonic acid (JA) is a signal transducer molecule that plays an important role in plant development and stress response; it can also efficiently stimulate secondary metabolism in plant cells. RESULTS: RNA-Seq technology was applied to identify differentially expressed genes and study the time course of gene expression in Rhazya stricta in response to JA. Of more than 288 million total reads, approximately 27% were mapped to genes in the reference genome. Genes involved during the secondary metabolite pathways were up- or downregulated when treated with JA in R. stricta. Functional annotation and pathway analysis of all up- and downregulated genes identified many biological processes and molecular functions. Jasmonic acid biosynthetic, cell wall organization, and chlorophyll metabolic processes were upregulated at days 2, 6, and 12, respectively. Similarly, the molecular functions of calcium-transporting ATPase activity, ADP binding, and protein kinase activity were also upregulated at days 2, 6, and 12, respectively. Time-dependent transcriptional gene expression analysis showed that JA can induce signaling in the phenylpropanoid and aromatic acid pathways. These pathways are responsible for the production of secondary metabolites, which are essential for the development and environmental defense mechanism of R. stricta during stress conditions. CONCLUSIONS: Our results suggested that genes involved in flavonoid biosynthesis and aromatic acid synthesis pathways were upregulated during JA stress. However, monoterpenoid indole alkaloid (MIA) was unaffected by JA treatment. Hence, we can postulate that JA plays an important role in R. stricta during plant development and environmental stress conditions.

Cyclopentanes/metabolism , Apocynaceae/genetics , Oxylipins/metabolism , Plant Growth Regulators/metabolism , Stress, Physiological , Flavonoids/biosynthesis , Base Sequence , Gene Expression , Environment , Transcriptome
Arq. bras. med. vet. zootec. (Online) ; 73(2): 302-310, Mar.-Apr. 2021. tab
Article in English | ID: biblio-1248934


Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.(AU)

A mastite clínica bovina causada por Staphylococcus spp. é uma doença grave e generalizada no mundo da pecuária leiteira. A terapia antimicrobiana é de fundamental importância na prevenção e no tratamento da mastite infecciosa, mas o uso indiscriminado de antimicrobianos atua como fator determinante para a disseminação da doença. O presente estudo avaliou os perfis de resistência de 57 Staphylococcus spp. isolados de mastite clínica bovina em relação ao uso de betalactâmicos e gentamicina, relacionando características do fenótipo (testes de suscetibilidade in vitro) e genótipo (detecção e expressão de genes que codificam resistência - mecA, mecALGA251, blaZ, femA, femB, e aacA-aphD - usando PCR e RT-PCR, respectivamente). Um ou mais genes que codificam resistência a diferentes antimicrobianos foram detectados em 50 Staphylococcus spp. isolados. Os genes femA e femB foram os mais frequentes (75,4% para ambos). A expressão observada dos genes foi a seguinte: blaZ (60%), femA (39,5%), aacA-aphD (50%), femB (32,6%), mecA (8,3%) e mecALGA251 (0%). Considerando-se a relevância do gênero Staphylococcus para a mastite bovina, este estudo teve como objetivo elucidar aspectos referentes aos perfis genotípico e fenotípico desses microrganismos, a fim de contribuir para o desenvolvimento de estratégias eficazes para o controle da mastite.(AU)

Staphylococcus/isolation & purification , Gene Expression/genetics , beta-Lactam Resistance/genetics , Drug Resistance, Bacterial/genetics , Mastitis, Bovine/microbiology , Gentamicins , Reverse Transcriptase Polymerase Chain Reaction
Article in Chinese | WPRIM | ID: wpr-880834


OBJECTIVE@#To explore the mechanism by which ginsenoside 20(S)-Rg3 upregulates the expression of tumor suppressor von Hippel-Lindau (VHL) gene in ovarian cancer cells.@*METHODS@#Ovarian cancer cell line SKOV3 treated with 20(S)-Rg3 were examined for mRNA and protein levels of VHL, DNMT1, DNMT3A and DNMT3B by real-time PCR and Western blotting, respectively. The changes in VHL mRNA expression in SKOV3 cells in response to treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, were detected using real-time PCR. VHL gene promoter methylation was examined with methylation-specific PCR and VHL expression levels were determined with real-time PCR and Western blotting in non-treated or 20(S)-Rg3-treated SKOV3 cells and in 20(S)-Rg3-treated DNMT3A-overexpressing SKOV3 cells. VHL and DNMT3A protein levels were detected by immunohistochemistry in subcutaneous SKOV3 cell xenografts in nude mice.@*RESULTS@#Treatment of SKOV3 cells with 20(S)-Rg3 significantly upregulated VHL and downregulated DNMT3A expressions at both the mRNA and protein levels (@*CONCLUSIONS@#Ginsenoside 20(S)-Rg3 upregulates VHL expression in ovarian cancer cells by suppressing DNMT3A-mediated DNA methylation.

Animals , Cell Line, Tumor , DNA Methylation , Female , Gene Expression , Ginsenosides/pharmacology , Humans , Mice , Mice, Nude , Ovarian Neoplasms/genetics , Promoter Regions, Genetic , Von Hippel-Lindau Tumor Suppressor Protein/genetics
Article in English | WPRIM | ID: wpr-880615


OBJECTIVES@#To study the gene expression of adipose tissue CD14@*METHODS@#The data of GSE54350 were obtained from the public database of gene expression profiling. The data were pre-processed by Network Analyst, String 11.0, Cytoscape 3.7.1, and other analytical software. The differentially expressed genes were analyzed by gene ontology biological function and kyoto encycopedia of genes and genomes (KEGG) signaling pathway to establish differential gene protein interaction network, transcription factor-gene regulatory network, microRNA-gene regulatory network, environmental factors-gene regulatory network, and other interaction systems.@*RESULTS@#The gene expression pattern of CD14@*CONCLUSIONS@#The gene expression of adipose tissue CD14

Adipose Tissue , Computational Biology , DNA-Binding Proteins , Diabetes Mellitus, Type 2/genetics , Gene Expression , Gene Expression Profiling , Gene Regulatory Networks , Humans , MicroRNAs/genetics , Muscle Proteins
Article in English | WPRIM | ID: wpr-880352


BACKGROUND@#Arsenic is a developmental neurotoxicant. It means that its neurotoxic effect could occur in offspring by maternal arsenic exposure. Our previous study showed that developmental arsenic exposure impaired social behavior and serotonergic system in C3H adult male mice. These effects might affect the next generation with no direct exposure to arsenic. This study aimed to detect the social behavior and related gene expression changes in F2 male mice born to gestationally arsenite-exposed F1 mice.@*METHODS@#Pregnant C3H/HeN mice (F0) were given free access to tap water (control mice) or tap water containing 85 ppm sodium arsenite from days 8 to 18 of gestation. Arsenite was not given to F1 or F2 mice. The F2 mice were generated by mating among control F1 males and females, and arsenite-F1 males and females at the age of 10 weeks. At 41 weeks and 74 weeks of age respectively, F2 males were used for the assessment of social behavior by a three-chamber social behavior apparatus. Histological features of the prefrontal cortex were studied by ordinary light microscope. Social behavior-related gene expressions were determined in the prefrontal cortex by real time RT-PCR method.@*RESULTS@#The arsenite-F2 male mice showed significantly poor sociability and social novelty preference in both 41-week-old group and 74-week-old group. There was no significant histological difference between the control mice and the arsenite-F2 mice. Regarding gene expression, serotonin receptor 5B (5-HT 5B) mRNA expression was significantly decreased (p < 0.05) in the arsenite-F2 male mice compared to the control F2 male mice in both groups. Brain-derived neurotrophic factor (BDNF) and dopamine receptor D1a (Drd1a) gene expressions were significantly decreased (p < 0.05) only in the arsenite-F2 male mice of the 74-week-old group. Heme oxygenase-1 (HO-1) gene expression was significantly increased (p < 0.001) in the arsenite-F2 male mice of both groups, but plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) and cyclooxygenase-2 (COX-2) gene expression were not significantly different. Interleukin-1β (IL-1β) mRNA expression was significantly increased only in 41-week-old arsenite-F2 mice.@*CONCLUSIONS@#These findings suggest that maternal arsenic exposure affects social behavior in F2 male mice via serotonergic system in the prefrontal cortex. In this study, COX-2 were not increased although oxidative stress marker (HO-1) was increased significantly in arsnite-F2 male mice.

Animals , Arsenic/toxicity , Arsenites/toxicity , Behavior, Animal/drug effects , Environmental Pollutants/toxicity , Female , Gene Expression/drug effects , Genetic Markers , Male , Maternal Exposure/adverse effects , Mice , Mice, Inbred C3H , Oxidative Stress/genetics , Prefrontal Cortex/drug effects , Pregnancy , Prenatal Exposure Delayed Effects/psychology , Reverse Transcriptase Polymerase Chain Reaction , Serotonin/metabolism , Social Behavior , Sodium Compounds/toxicity
Article in Chinese | WPRIM | ID: wpr-878987


To investigate the effect of Gegen Qinlian Decoction(GQD) on enzyme activity, gene expression and methylation level of fatty acid synthase(FASN) in adipose tissue from rats with insulin resistance induced by high-fat diet. The 60% fat-powered high-fat diet was continuously given to male SD rats to induce the insulin resistance model. Then, they were divided into five groups randomly and administrated by gavage every day for 16 weeks with following drugs respectively: 10 mL·kg~(-1)water for control group(C) and insulin resistance model control group(IR), 1.65 g·kg~(-1)GQD per day for low-dose group(GQDL), 4.95 g·kg~(-1)GQD per day for medium-dose group(GQDM), 14.85 g·kg~(-1)GQD per day for high-dose group(GQDH), and 5 mg·kg~(-1) rosiglitazone per day for rosiglitazone group(RGN). Epididymal adipose tissue was taken to determine enzyme activity of FASN by colorimetric method, mRNA expression level of Fasn by quantitative Real-time PCR(Q-PCR) and CpGs methylation level between +313 and +582 by bisulfite sequencing PCR(BSP). These results showed that Fasn expression was significantly lowered in IR model rats compared with the control rats(P<0.01). Enzymatic activity and CpGs methylation level of Fasn in IR group showed downward trends. Low and medium-dose GQD can increase enzyme activity of FASN(P<0.05). Moreover, low-dose GQD increased the total CpGs methylation level of Fasn fragment between +313 and +582 in insulin resistance rats(P<0.05). For GQDM group, the methylation frequency of CpGs at positions +506 and +508(P<0.01) as well as the methylation frequency of CpGs on the binding sites of transcription factorzinc finger protein 161(P<0.05) were significantly increased. The methylation frequency of CpG at +442 position was positively correlated with Fasn expression(P<0.01, r=0.735), and methylation frequencies of CpGs at +345 and +366 positions were positively associated to enzyme activity of FASN respectively(P<0.05, r=0.479; P<0.01, r=0.640). In conclusion, GQD can reverse enzyme activity of FASN and methylation level of Fasn in adipose tissue of insulin resistant rats, and CpG sites at positions +506 and +508 may be the targets of GQD. The methylation level of CpGs at + 345 and + 366 sites were possibly related to FASN activity, while methylation of CpG at + 442 site may be closely correlated with mRNA level of Fasn. In addition, GQD did not significantly change mRNA expression level of Fasn, but effectively reversed enzymatic activity, suggesting that GQD may regulate the post transcriptional expression of Fasn.

Adipose Tissue , Animals , Drugs, Chinese Herbal , Fatty Acid Synthases/genetics , Gene Expression , Insulin Resistance/genetics , Male , Methylation , Rats , Rats, Sprague-Dawley
Article in Chinese | WPRIM | ID: wpr-878730


In eukaryote cells,transcription from genome DNA is a key process of gene expression.The transcription products contain not only messenger RNAs that code proteins,but also various types of non-coding RNAs.During transcription,some of the gene loci produce more than one kind of RNA molecule,including coding RNAs and more often non-coding RNAs.These gene loci that generate several kinds of RNA molecules are named supergenes.According to the transcription pattern,supergenes are divided into three types,known as types Ⅰ,Ⅱ and Ⅲ.In this review,we summarize the transcription pattern of each type of supergene,and exposit the role of these genes in cells.

Gene Expression , RNA, Messenger
Chinese Journal of Biotechnology ; (12): 655-662, 2021.
Article in Chinese | WPRIM | ID: wpr-878590


The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.

Animals , Animals, Genetically Modified , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Houseflies/genetics , Microinjections
Chinese Medical Journal ; (24): 935-943, 2021.
Article in English | WPRIM | ID: wpr-878142


BACKGROUND@#Since 2019, a novel coronavirus named 2019 novel coronavirus (2019-nCoV) has emerged worldwide. Apart from fever and respiratory complications, acute kidney injury has been observed in a few patients with coronavirus disease 2019. Furthermore, according to recent findings, the virus has been detected in urine. Angiotensin-converting enzyme II (ACE2) has been proposed to serve as the receptor for the entry of 2019-nCoV, which is the same as that for the severe acute respiratory syndrome. This study aimed to investigate the possible cause of kidney damage and the potential route of 2019-nCoV infection in the urinary system.@*METHODS@#We used both published kidney and bladder cell atlas data and new independent kidney single-cell RNA sequencing data generated in-house to evaluate ACE2 gene expression in all cell types in healthy kidneys and bladders. The Pearson correlation coefficients between ACE2 and all other genes were first generated. Then, genes with r values larger than 0.1 and P values smaller than 0.01 were deemed significant co-expression genes with ACE2.@*RESULTS@#Our results showed the enriched expression of ACE2 in all subtypes of proximal tubule (PT) cells of the kidney. ACE2 expression was found in 5.12%, 5.80%, and 14.38% of the proximal convoluted tubule cells, PT cells, and proximal straight tubule cells, respectively, in three published kidney cell atlas datasets. In addition, ACE2 expression was also confirmed in 12.05%, 6.80%, and 10.20% of cells of the proximal convoluted tubule, PT, and proximal straight tubule, respectively, in our own two healthy kidney samples. For the analysis of public data from three bladder samples, ACE2 expression was low but detectable in bladder epithelial cells. Only 0.25% and 1.28% of intermediate cells and umbrella cells, respectively, had ACE2 expression.@*CONCLUSION@#This study has provided bioinformatics evidence of the potential route of 2019-nCoV infection in the urinary system.

Angiotensin-Converting Enzyme 2/metabolism , COVID-19 , Gene Expression , Humans , Kidney/metabolism , SARS-CoV-2 , Sequence Analysis, RNA , Single-Cell Analysis , Urinary Bladder/metabolism
Clinics ; 76: e2846, 2021. tab, graf
Article in English | LILACS | ID: biblio-1278914


Breast cancer is the most frequently diagnosed malignant neoplasm in women and is considered a multifactorial disease of unknown etiology. One of the major risk factors is genetic alteration. Changes in CYP19A1 gene expression levels have been associated with increased risk and increased aggressiveness of breast cancer. Increased CYP19A1 gene expression and/or aromatase activity are among the major regulatory events for intratumoral production of estrogens in breast malignant tissues. This systematic review aimed to investigate the influence of CYP19A1 gene expression levels in women with breast cancer. The research was carried out using the PubMed, Scopus, and Web of Science databases. Searches were conducted between February 2 and May 15, 2019. Inclusion criteria were studies published between 2009 and 2019, English language publications, and human studies addressing the gene expression of CYP19A1 in breast cancer. A total of 6.068 studies were identified through PubMed (n=773), Scopus (n=2,927), and the Web of Science (n=2,368). After selecting and applying the inclusion and exclusion criteria, six articles were included in this systematic review. This systematic review provides evidence that increased or decreased levels of CYP19A1 gene expression may be related to pathological clinical factors of disease, MFS, OS, DFS, WATi, markers of metabolic function, concentrations of E1, FSH, and in the use of multiple exons 1 of the CYP19A1 gene in breast cancer.

Humans , Female , Breast Neoplasms/genetics , RNA, Messenger , Aromatase/genetics , Gene Expression , Estrogens
Braz. arch. biol. technol ; 64: e21210292, 2021. tab, graf
Article in English | LILACS | ID: biblio-1278439


Abstract NADPH-cytochromeP450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. In this study, a gene encoding CPR (named EsCPR) was isolated from Eriocheir sinensis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. Analysis of the nucleotide sequence revealed a cDNA full-length of 3717 bp with an open reading frame of 2046 bp, a 5′-untranslated region of 42 bp, and a long 3′-untryganslated region of 1628bp, which encodes a protein of 681 amino acids with a predicted molecular weight of 30.7 kDa and an estimated pI of 4.82. The mature peptide shares amino acid of E. sinensis identity 82 % - 89 % to the CPR from Penaeus vannamei and Chionoecetes opilio. Tissues and developmental stage-dependent expression of EsCPR mRNA was investigated by real-time quantitative PCR. EsCPR mRNA was markedly expressed in the hepatopancreas and stomach. These results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems.

NADPH-Ferrihemoprotein Reductase , Cloning, Organism , Brachyura , Gene Expression , Reverse Transcriptase Polymerase Chain Reaction
São Paulo; s.n; 2021. 92 p. ilust, tabelas.
Thesis in Portuguese | LILACS, Inca | ID: biblio-1223738


Mutações somáticas não sinônimas podem iniciar a tumorigênese e, também, uma resposta citotóxica antitumoral. Com o desenvolvimento das tecnologias de sequenciamento, tornou-se possível identificar as mutações em todos os genes humanos e, consequentemente, as variantes que induzem uma resposta imune (neoantígenos), representando uma oportunidade para pacientes que possam se beneficiar de imunoterapias, mas também um desafio com a necessidade de várias camadas de informações e a integração computacional de vários tipos de dados. Neste trabalho, foi desenvolvido o pipeline de identificação de neoantígeno neo2P, o qual realiza a integração completa de todos os passos necessários para a detecção e neoantígenos e apresentou uma eficiência computacional superior de até seis vezes em comparação com outro método. Além disso, foi proposto um score para priorizaração das mutações somáticas a partir da distribuição dos níveis da expressão gênica de 9.679 pacientes de 32 projetos do TCGA, o qual apresentou um poder de discriminação (AUC) próximo ou superior a 0.7 na maioria das coortes avaliadas. O neo2P foi aplicado em um conjunto de dados de pacientes com melanoma e foram identificados aspectos adicionais da relação de neoantígenos e aspectos imunes, como a expressão de alguns genes marcadores que podem estar relacionados com a resposta ao tratamento. Adicionalmente, a carga de neoantígenos detectados pelo neo2P estratificou, de maneira significativa, pacientes respondedores (R) e não respondedores (NR) quando comparado com o marcador TMB

Somatic non-synonymous mutations can initiate tumorigenesis and, conversely, anti-tumor cytotoxic T cell (CTL) responses. With the development of next-generation sequencing, it has become feasible to detect mutation-derived neoantigens within exome and thereby predict potential neoantigens, which represents an opportunity to patients that may be treated with immunotherapies, but also a challenge due to multiple layers of information and a computational integration of several types of data. In this work, it was developed a neoantigen identification pipeline called neo2P, which integrates all the necessary steps involved for neoantigen detection and presented a six-times superior computational efficiency compared to another method. In addition, a score was proposed to prioritize somatic mutations based on the distribution of gene expression levels in 9,679 patients from 32 TCGA projects, which showed a stratification ability (AUC) close to or greater than 0.7 in most evaluated cohorts.neo2P was applied to a dataset of patients with melanoma and additional aspects of the relationship between neoantigens and immune aspects were identified, such as the expression of some marker genes that may be related to the treatment response. Additionally, the neoantigen load detected by neo2P significantly stratified responders (R) and non-responders (NR) patients when compared to the TMB marker

Prognosis , Gene Expression , Computational Biology , Immunotherapy , Melanoma
Einstein (Säo Paulo) ; 19: eRB5996, 2021. tab, graf
Article in English | LILACS | ID: biblio-1286282


ABSTRACT MicroRNAs are small RNA molecules that regulate gene expression in cells. These small molecules comprise 17 to 25 nucleotides and are able to recognize target messenger RNAs by sequence complementarity and regulate their protein translation. Different microRNAs are expressed in all human cells. There is over 2,500 microRNAs described in humans that are involved in virtually all biological processes. Given their role as gene expression regulators, these molecules have been widely investigated and are thought to be associated with some specific physiological and pathological conditions, being proposed as biomarkers. It has recently been reported that microRNAs are secreted outside cells and are involved in intercellular communication. MicroRNAs in biological fluids are named circulating and have been detected in all body fluids, although the expression profile is specific for each type. The major advantages of using circulating microRNAs as biological markers are the high stability of those molecules and the wide availability of samples. Also, given the individual nature of microRNA expression changes, these molecules have a high potential for use in personalized medicine. In fact, microRNA expression profile determination may support disease recognition and diagnosis, and can be used to monitor therapeutic responses and establish patient prognosis, assisting in choice of treatment. This review provides a general overview of microRNAs and discusses the importance of those molecules in cancer, for deeper understanding of their role in this disease.

RESUMO Os microRNAs são pequenas moléculas de RNAs que regulam a expressão gênica das células. Com entre 17 e 25 nucleotídeos, essas pequenas moléculas reconhecem RNA mensageiro-alvo, por meio da complementariedade entre as sequências, e regulam sua tradução proteica. Todas as células humanas expressam diversos microRNAs. De fato, existem mais de 2.500 microRNAs descritos em humanos, relacionados com praticamente todos os processos biológicos. Devido ao seu papel como reguladores da expressão gênica, essas moléculas têm sido estudadas e relacionadas com algumas condições fisiológicas e patológicas específicas, sendo propostas como biomarcadores. Recentemente, foi descoberto que os microRNAs são normalmente liberados para fora da célula, onde participam da comunicação intercelular. MicroRNAs presentes nos fluidos biológicos são chamados de circulantes e têm sido encontrados em todos os fluidos corporais, porém o perfil de expressão é específico para cada tipo. O uso de microRNAs circulantes como marcadores biológicos apresenta vantagens relacionadas com a alta estabilidade dessas moléculas e a facilidade de obtenção de amostra. Adicionalmente, considerando que as alterações em microRNAs são dependentes das condições individuais, essas moléculas apresentam alto potencial de uso na medicina personalizada. De fato, a determinação do perfil de expressão de microRNAs pode auxiliar na identificação e diagnóstico de doenças, no monitoramento de respostas terapêuticas e na definição do prognóstico dos pacientes, auxiliando na escolha do tratamento. Nesta revisão são apresentados aspectos gerais dos microRNAs, e discute-se a importância dessas moléculas no câncer, visando a uma melhor compreensão de seu papel nessa doença.

Humans , MicroRNAs/genetics , Neoplasms/genetics , Prognosis , Biomarkers , Gene Expression
Mem. Inst. Oswaldo Cruz ; 116: e200592, 2021. tab, graf
Article in English | LILACS | ID: biblio-1154883


BACKGROUND Paracoccidioidomycosis (PCM) is a systemic mycosis with high prevalence in Latin America that is caused by thermodimorphic fungal species of the Paracoccidioides genus. OBJECTIVES In this study, we used quantitative polymerase chain reaction (qPCR) to investigate the expression of genes related to the virulence of Paracoccidioides brasiliensis (Pb18) and P. lutzii (Pb01) strains in their mycelial (M) and yeast (Y) forms after contact with alveolar macrophages (AMJ2-C11 cell line) and fibroblasts (MRC-5 cell line). METHODS The selected genes were those coding for 43 kDa glycoprotein (gp43), enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 14-3-3 protein (30 kDa), phospholipase, and aspartyl protease. FINDINGS In the Pb18 M form, the aspartyl protease gene showed the highest expression among all genes tested, both before and after infection of host cells. In the Pb18 Y form after macrophage infection, the 14-3-3 gene showed the highest expression among all genes tested, followed by the phospholipase and gp43 genes, and their expression was 50-fold, 10-fold, and 6-fold higher, respectively, than that in the M form. After fibroblast infection with the Pb18 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 25-fold, 10-fold, and 10-fold higher, respectively, than that in the M form. Enolase and aspartyl protease genes were expressed upon infection of both cell lines. After macrophage infection with the Pb01 Y form, the 14-3-3 gene showed the highest expression, followed by the phospholipase and aspartyl protease genes, and their expression was 18-fold, 12.5-fold, and 6-fold higher, respectively, than that in the M form. MAIN CONCLUSIONS In conclusion, the data show that the expression of the genes analysed may be upregulated upon fungus-host interaction. Therefore, these genes may be involved in the pathogenesis of paracoccidioidomycosis.

Humans , Paracoccidioides/genetics , Paracoccidioidomycosis/genetics , Virulence Factors/genetics , Fibroblasts , Macrophages , Paracoccidioides/pathogenicity , Gene Expression , Latin America