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1.
Medicine (Baltimore) ; 98(38): e17225, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31567982

RESUMO

The present study is to analyze the difference of gene methylation in early cervical adenocarcinoma and to find molecular markers for predicting the occurrence and development of cervical adenocarcinoma.A total of 15 cases of primary cervical adenocarcinoma and 10 cases of primary cervical squamous cell carcinoma at stages IB1 or IIA1 were included in the study. Infinium MethylationEPIC BeadChip (850K) was used to screen specifically expressed genes in cervical adenocarcinoma tissues. Bisulfite sequencing polymerase chain reaction (BSP) and quantitative real-time polymerase chain reaction (qRT-PCR) were used to verify the methylation levels in cervical adenocarcinoma, cervical squamous cell carcinoma, and normal cervical tissues.Sex determining region Y-box 1 (SOX1) and cyclin D1 (CCND1) genes participated in multiple signaling pathways, being the central nodes of gene regulatory networks. SOX1 gene, but not CCND1 gene, was a specifically methylated gene in cervical adenocarcinoma according to BSP. According to qRT-PCR, methylation level of SOX1 in cervical adenocarcinoma tissues is significantly different from that in cervical squamous cell carcinoma tissues or normal cervical tissues, and the methylation level of CCND1 in cervical adenocarcinoma tissues or cervical squamous cell carcinoma tissues is significantly different from that in normal cervical tissues.The present study demonstrates that tumor-suppressor gene SOX1 is a methylation-specific expression gene of cervical adenocarcinoma and is expected to become a specific molecular marker for the diagnosis of cervical adenocarcinoma. However, CCND1 gene was not proven to be a specific methylation expression gene in cervical adenocarcinoma in the present study.


Assuntos
Adenocarcinoma/genética , Metilação de DNA/genética , Fatores de Transcrição SOXB1/genética , Neoplasias do Colo do Útero/genética , Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Colo do Útero/metabolismo , Ciclina D1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes/genética , Marcadores Genéticos/genética , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias do Colo do Útero/metabolismo
2.
Exp Suppl ; 111: 33-52, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31588527

RESUMO

Molecular genetic methods have become an organic part of everyday clinical practice. In the past, molecular diagnostic tests were carried out for genetic diagnosis of a particular monogenic disease. In these situations the tests itself were used for identification of one particular genetic alteration (e.g., point mutation or deletion) of the gene of interest. Later, parallel with the development of the technology, the focus has shifted by allowing investigating at once targeted gene panels and even the whole exome/genome behind a suspected genetic disorder. Historically for these purposes, array-based methods (oligonucleotide arrays) and then next-generation sequencing-based methods have been used. High-throughput methods have been fundamentally transforming the everyday, routine genetic diagnostics, but older molecular techniques still have a role in clinical genetics. Here, we summarize the most important molecular genetic methods and shed light to the advantages and disadvantages of their application in routine diagnostics. We mainly focus on methods used for detection of germline alterations.


Assuntos
Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase em Tempo Real , Exoma , Humanos , Mutação
3.
Biol Res ; 52(1): 52, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31540582

RESUMO

BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to be associated with dermis process during burn wound healing. This study aimed to investigate the role of lncRNA X-inactive specific transcript (XIST) in human skin fibroblasts (HSF) and extracellular matrix (ECM) as well as the regulatory network of XIST/microRNA-29b-3p (miR-29b-3p)/collagen 1 alpha 1 (COL1A1). METHODS: The wound samples were collected from 25 patients with deep partial thickness burn at day 5 after burn. The thermal injured model was established using HSF cells. The expressions of XIST, miR-29b-3p and COL1A1 were measured by quantitative real-time polymerase chain reaction and western blot. ECM synthesis, cell proliferation and migration were detected by western blot, cell counting kit-8 and trans-well assays, respectively. The interaction between miR-29b-3p and XIST or COL1A1 was explored by bioinformatics analysis and luciferase reporter assay. RESULTS: The expressions of XIST and COL1A1 were enhanced but miR-29b-3p expression was decreased after thermal injury. XIST overexpression promoted ECM synthesis, cell proliferation and migration in thermal injured HSF cells. However, XIST knockdown played an opposite effect. miR-29b-3p overexpression inhibited ECM synthesis, cell proliferation and migration, which was reversed by XIST. COL1A1 silence suppressed ECM synthesis, cell proliferation and migration by miR-29b-3p targeting. Moreover, COL1A1 up-regulation weakened the effect of XIST silence on ECM synthesis and HSF cell function. CONCLUSION: XIST promoted ECM synthesis, cell proliferation and migration by sponging miR-29b-3p and targeting COL1A1 in HSF cells after thermal injury, indicating the promoting role of XIST in wound healing.


Assuntos
Queimaduras/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , RNA Longo não Codificante/metabolismo , Western Blotting , Queimaduras/genética , Movimento Celular , Proliferação de Células , Matriz Extracelular/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Reação em Cadeia da Polimerase em Tempo Real
4.
Rev Soc Bras Med Trop ; 52: e20190171, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31508782

RESUMO

INTRODUCTION: Biomphalaria glabrata is considered to be responsible for the incidence of schistosomiasis in Brazil. Therefore, surveillance of areas where schistosomiasis is prevalent is fundamental for public health planning. This study was aimed to evaluate B. glabrata populations in water bodies of the city of Salvador, determine their distribution, estimate the prevalence of Schistosoma mansoni infections, characterize shed cercariae, and identify transmission foci. METHODS: Malacological surveys were carried out in 17 water collections from Salvador. Snail species were identified based on shell and mantle characteristics. Snails were evaluated for S. mansoni infection by exposure to light and via real time polymerase chain reaction (qPCR) using S. mansoni-18S rRNA subunit specific primers. RESULTS: 1,403 B. glabrata were collected. Classical cercarial shedding indicated that 5 snails (0.4%) were positive for S. mansoni. A higher prevalence of infections was found in Horta de Saramandaia (5.5%) and Lagoa do IAT (1.9%). Non-Schistosoma larvae, such as Xiphidiocercaria, Strigeidae, Spirorchiidae and Clinostomidae, were observed in 3.2% of the snails. S. mansoni DNA was detected in 6.2% snails via qPCR. CONCLUSIONS: B. glabrata is widely distributed in Salvador, as indicated by 7 water collections associated with a risk of schistosomiasis transmission. To our knowledge, this is the first study to identify B. glabrata eliminating cercariae of Clinostomidae, Strigeidae, and Spirorchiidae in Salvador. We propose that qPCR may be employed in combination with classical cercarial shedding. Estimating S. mansoni prevalence in snails by only considering the results of light exposure method classical into account may underestimate the problem.


Assuntos
Biomphalaria/parasitologia , Vetores de Doenças , Schistosoma mansoni/genética , Animais , Humanos , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Schistosoma mansoni/isolamento & purificação , Esquistossomose mansoni/transmissão , População Urbana
5.
Fa Yi Xue Za Zhi ; 35(4): 387-392, 2019 Aug.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-31532143

RESUMO

Abstract: Objective Quantitative analysis and comparison of the expression of ribonucleic acid (RNA) from frozen organs and formaldehyde-fixed and paraffin-embedded (FFPE) tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin (ACTB) with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA (5S rRNA) achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs (miRNAs), GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.


Assuntos
Formaldeído , MicroRNAs/análise , Inclusão em Parafina , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real/normas , Primers do DNA , Perfilação da Expressão Gênica , Humanos , Miocárdio
6.
Fa Yi Xue Za Zhi ; 35(4): 396-401, 2019 Aug.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-31532145

RESUMO

Abstract: Objective To study the protein expression of cluster of differentiation 63 (CD63) in lung tissues of guinea pigs that died of anaphylactic shock and discuss the diagnostic value of CD63 for death from anaphylactic shock. Methods Twenty guinea pigs were randomly divided into control group, anaphylactic shock immediate death group, cold storage group (4 ℃ for 48 h) and frozen group (-20 ℃ for 7 d). The animal model of guinea pigs that died of anaphylactic shock was established with human mixed serum injection. The expression changes of CD63 protein and CD63 mRNA in lung tissues were detected by hematoxylin-eosin (HE) staining, immunohistochemical staining, Western blotting, enzyme-linked immunosorbent assay (ELISA) and real-time RT-PCR. Results HE staining results showed congestion, and edema of lung tissues, and eosinophil infiltration in the anaphylactic shock groups. Western blotting analysis results showed that the expression of CD63 protein in the lung tissues of guinea pigs that died of anaphylactic shock was significantly higher than that in the control group (P<0.05). Comparison between the anaphylactic shock groups was made, and the differences had no statistical significance. The results of immunohistochemical staining and real-time RT-PCR were consistent with that of Western blotting. ELISA results showed that CD63 protein expression in the immediate death group was higher than that in the control group (P<0.05). Conclusion The expression of CD63 protein and CD63 mRNA in the lung tissues of guinea pigs that died of anaphylactic shock is significantly enhanced. Animal carcasses which were put in cold storage for 48 h and frozen for 7 d do not affect the examination of the above indicators. CD63 protein is expected to become an auxiliary diagnostic indicator of death from anaphylactic shock.


Assuntos
Anafilaxia/metabolismo , Pulmão/metabolismo , Tetraspanina 30/metabolismo , Anafilaxia/mortalidade , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Cobaias , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro
7.
Medicina (B Aires) ; 79(4): 265-270, 2019.
Artigo em Espanhol | MEDLINE | ID: mdl-31487245

RESUMO

Malignant melanoma (MM) is the more aggressive form of skin cancer with a mortality rate in Argentina 1997-2001 = 1.1/100 000 in men and 0.6 in women. BRAF proto-oncogene is focus of intense research; its mutation is one of the main tumor promoters and occurs in approximately 50% of MM. Several drugs with clinical activity on BRAF mutations have been approved. The aim of the study is to evaluate the mutational status of BRAF (exon 15) in cutaneous MM biopsies and its relationship with histopathological characteristics. We carried out an observational, retrospective study of samples fixed in formaldehyde and paraffin embedded; reviewing age, sex, diagnosis, histopathological data, tumor size and percentage, viability for molecular analysis and melanin presence. We evaluated BRAF mutations with PCR/Sanger sequencing. For statistics we used Student's t test, Chi square, Wilcoxon and Fisher's exact test. We were able to purify and sequence 76% (38/49) samples, 13/38 (34%) from women and 25/38 (66%) from men, the median age being 70 years. Most frequent location: thorax 14/35 (40%). Histological type: Superficial spreading 18/38 (47%). Clark's levels, 11/38 (29%): I-II and 27/38 (71%): III, IV and V. Breslow's median: 1.6 mm. Radial growth phase 11/38 (29%) and 27/38 (71%) vertical. Presented mutations 16/38 (42%). As reported by other authors, no association was found between the mutational state of exon 15 and clinical or histopathological parameters.


Assuntos
Melanoma/genética , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Melanoma/patologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Neoplasias Cutâneas/patologia
8.
Klin Lab Diagn ; 64(8): 493-496, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31479606

RESUMO

The aim of the work was to comparison of rayon and flocked swabs for collection and transport of deep throat swabs for detection of bacteria causing whooping cough by multiplex real-time PCR assay. The study included 87 patients aged from 1 month to 37 years, hospitalized in Infectious Diseases Clinical Hospital No. 1 of the Moscow Department of Healthcare. 68 (78,2 %) people had a diagnosis of whooping cough, the main group of which consisted of children aged 1 to 12 months (median 4 months); 17 (19,5 %) - other diseases of the respiratory tract; 2 (2,3 %) - contact with sick whooping cough. The initial examination of patients was carried out on the 1 - 8th week of the onset of the disease. The material from the patients was taken at one-day interval with commercial rayon swabs and flocked swabs. Identification and differentiation of specific genome fragments of the causative agents of whooping cough in biological material was carried out by real-time PCR using the «AmpliSens® Bordetella multi-FL¼ reagent kit. The efficiency of PCR-based diagnostics of whooping cough using flocked swabs at the preanalytical stage was 83,8 %, and rayon swabs - 82,3 %. The use of a flocked swabs at the preanalytical stage increased the research efficiency by 1,5 %. Thus, when collecting biological material for PCR-based diagnostics of whooping cough it is possible to use flocked swabs.


Assuntos
Celulose , Manejo de Espécimes/instrumentação , Coqueluche/diagnóstico , Adolescente , Adulto , Bactérias , Criança , Pré-Escolar , Humanos , Lactente , Moscou , Faringe , Reação em Cadeia da Polimerase em Tempo Real , Adulto Jovem
9.
Zhonghua Fu Chan Ke Za Zhi ; 54(9): 601-607, 2019 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-31550776

RESUMO

Objective: To explore the pathways of preeclampsia by investigating different effects of pravastatin (Pra) on and soluble FMS tyrosine kinase-1 (sFlt-1), placental growth factor (PlGF) and vascular endothelial growth factor (VEGF) in different preeclampsia (PE)-like mouse models. Methods: C57BL/6J mice were randomly subcutaneously injected with N-nitro-L-arginine methyl ester (L-NAME) or intraperitoneally injected with lipopolysaccharide (LPS) as PE-like mouse model, saline as normal pregnancy control (Con) respectively, daily at gestational 7-18 days. Pra was given daily at gestational 8-18 days in each model group and the mice were divided into Pra (L-NAME+Pra, LPS+Pra, Con+Pra) and saline (L-NAME+NS, LPS+NS, Con+NS) groups. Liver,placental tissue and blood of pregnant mice were collected on the 18th day of pregnancy. The levels of VEGF, PlGF and sFlt-1 in the liver, placenta and serum of mice in each group were compared by western blot, ELISA and real-time fluorescence quantitative PCR (RT-PCR). Results: (1) ELISA: Serum VEGF (205.70±3.43, 154.60±2.31) and PlGF (131.5±3.75, 101.50±4.31) levels were significantly increased in L-NAME+Pra group compared with L-NAME+NS group (all P<0.05). Serum VEGF (202.30±4.90, 144.50±6.71) and PlGF (121.50±3.86, 95.41±4.08) levels were significantly higher in LPS+Pra group than those in LPS+NS group (all P<0.05). Serum sFlt-1 level in LPS+Pra group was significantly lower than that in LPS+NS group (3.01±0.50, 776.60±80.06), serum sFlt-1 level in L-NAME+Pra group was significantly lower than that in L-NAME+NS group (2.60±0.06, 583.70±9.83; all P<0.05). (2) Western blot: the expression levels of PlGF (1.344±0.118, 0.664±0.143) and VEGF (1.34±0.12, 0.66±0.14) in the liver of mice in the L-NAME+Pra group were significantly higher than those in the L-NAME+NS group (all P<0.05), but the expression levels of PlGF and VEGF in the placenta of L-NAME+Pra group were not significantly different from those of L-NAME+NS group (all P>0.05). The expression levels of PlGF and VEGF in placenta and liver of pregnant mice in LPS+Pra group were not significantly different from those in LPS+N group (all P>0.05). (3) RT-PCR: the mRNA expression of PlGF and VEGF in placenta and liver of L-NAME+Pra group were not significantly different from those in L-NAME+NS group (all P>0.05). The mRNA expression levels of PlGF and VEGF in placenta and liver of LPS+Pra group were not significantly different from those of LPS+NS group (all P>0.05). Conclusions: Pra has different regulatory effects on vascular endothelial function in different PE-like models. It reveals that different pathogenesis and pathways exist in different PE-like changes.


Assuntos
Fator de Crescimento Placentário/efeitos dos fármacos , Pravastatina/uso terapêutico , Pré-Eclâmpsia/tratamento farmacológico , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Animais , Anticolesterolemiantes/farmacologia , Biomarcadores/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Placenta , Fator de Crescimento Placentário/sangue , Reação em Cadeia da Polimerase , Pravastatina/farmacologia , Pré-Eclâmpsia/sangue , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/sangue , Fator A de Crescimento do Endotélio Vascular/sangue
10.
J Appl Oral Sci ; 27: e20180671, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31508795

RESUMO

OBJECTIVE: To monitor early periodontal disease progression and to investigate clinical and molecular profile of inflamed sites by means of crevicular fluid and gingival biopsy analysis. METHODOLOGY: Eighty-one samples of twenty-seven periodontitis subjects and periodontally healthy individuals were collected for the study. Measurements of clinical parameters were recorded at day -15, baseline and 2 months after basic periodontal treatment aiming at monitoring early variations ofthe clinical attachment level. Saliva, crevicular fluid and gingival biopsies were harvested from clinically inflamed and non-inflamed sites from periodontal patients and from control sites of healthy patients for the assessment of IL-10, MMP-8, VEGF, RANKL, OPG and TGF-ß1 protein and gene expression levels. RESULTS: Baseline IL-10 protein levels from inflamed sites were higher in comparison to both non-inflamed and control sites (p<0.05). Higher expression of mRNA for IL-10, RANK-L, OPG, e TGF-ß1 were also observed in inflamed sites at day -15 prior treatment (p<0.05). After the periodontal treatment and the resolution of inflammation, seventeen percent of evaluated sites still showed clinically detectable attachment loss without significant differences in the molecular profile. CONCLUSIONS: Clinical attachment loss is a negative event that may occur even after successful basic periodontal therapy, but it is small and limited to a small percentage of sites. Elevated inflammation markers of inflamed sites from disease patients reduced to the mean levels of those observed in healthy subjects after successful basic periodontal therapy. Significantly elevated both gene and protein levels of IL-10 in inflamed sites prior treatment confirms its modulatory role in the disease status.


Assuntos
Perda da Inserção Periodontal/patologia , Periodontite/patologia , Adulto , Biomarcadores/análise , Biópsia , Estudos de Casos e Controles , Citocinas/análise , Feminino , Gengiva/patologia , Líquido do Sulco Gengival/química , Humanos , Masculino , Metaloproteinase 8 da Matriz/análise , Pessoa de Meia-Idade , Osteoprotegerina/análise , Periodontite/terapia , Reação em Cadeia da Polimerase em Tempo Real , Saliva/química , Estatísticas não Paramétricas , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/análise
11.
Braz Oral Res ; 33: e0045, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31531560

RESUMO

The aim of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of dental follicle cells (DFCs) in vitro and on the regenerative effects of DFC-OsteoBoneTM complexes in vivo. DFCs were isolated and characterized. In the in vitro study, DFCs were cultured in an osteogenic medium in the presence or absence of LIPUS. The expression levels of ALP, Runx2, OSX, and COL-I mRNA were analyzed using real-time polymerase chain reaction (RT-PCR) on day 7. Alizarin red staining was performed on day 21. The state of the growth of the DFCs that were seeded on the scaffold at 3, 5, 7, and 9 days was detected by using a scanning electron microscope. In our in vivo study, 9 healthy nude mice randomly underwent subcutaneous transplantation surgery in one of three groups: group A, empty scaffold; group B, DFCs + scaffold; and group C, DFCs + scaffold + LIPUS. After 8 weeks of implantation, a histological analysis was performed by HE and Mason staining. Our results indicate that LIPUS promotes the osteogenic differentiation of DFCs by increasing the expression of the ALP, Runx2, OSX, and COL-I genes and the formation of mineralized nodules. The cells can adhere and grow on the scaffolds and grow best at 9 days. The HE and Mason staining results showed that more cells, fibrous tissue and blood vessels could be observed in the DFCs + scaffold + LIPUS group than in the other groups. LIPUS could promote the osteogenic differentiation of DFCs in vitro and promote tissue regeneration in a DFCs-scaffold complex in vivo. Further studies should be conducted to explore the underlying mechanisms of LIPUS.


Assuntos
Regeneração Óssea/efeitos da radiação , Saco Dentário/citologia , Osteogênese/efeitos da radiação , Terapia por Ultrassom/métodos , Ondas Ultrassônicas , Animais , Cerâmica , Saco Dentário/efeitos da radiação , Citometria de Fluxo , Camundongos Nus , Microscopia Eletrônica de Varredura , Distribuição Aleatória , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de Tempo
12.
Braz Oral Res ; 33: e077, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31531564

RESUMO

The aim of the present study was to identify the relationship between the expression of calcitonin gene-related peptide (CGRP) and the responses of pulp sensitivity tests in healthy pulps and irreversible pulps by performing a cross-sectional study on patients. Two hundred subjects were evaluated. A total of 75 subjects complied with the criteria. The participants were divided into two groups: a) Healthy pulp (subjects [n = 35] having posterior teeth with clinically normal pulp tissue), and b) Irreversible pulpitis (subjects [n = 40] having posterior teeth with irreversible pulpitis). All participants were evaluated using the following variables: a) medical and dental history, b) pulp sensitivity tests, c) expression of CGRP by the enzyme-linked immunosorbent assay (ELISA), and d) expression levels of mRNA CGRP and mRNA CGRP receptor genes. We determined that the responses of the cold test between 4 and ≥12 s presented a higher average of the expression of CGRP in the group having irreversible pulpitis (p = 0.0001). When we compared the groups with the value of the electrical impulse, we found statistically significant differences (p = 0.0001), observing positive responses to the test with electrical impulses of 7 to 10, with an average of 72.15 ng/mL of CGRP in the irreversible pulpitis group. High values of CGRP expression were observed in that group in the responses of pulp sensitivity.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/análise , Teste da Polpa Dentária/métodos , Polpa Dentária/patologia , Pulpite/patologia , Adolescente , Adulto , Estudos de Casos e Controles , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pulpite/genética , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Estatísticas não Paramétricas , Fatores de Tempo , Adulto Jovem
13.
Medicine (Baltimore) ; 98(35): e16970, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464941

RESUMO

This study aimed to determine the feasibility of vaginal/cervical nurse-assisted self-sampling (NASS) and the agreement between human papilloma virus (HPV) tests on self-samples versus clinician-taken (CT) specimens.Women participated voluntarily for cervical cancer screening at St. Aklesia Memorial Hospital. Eighty-three women provided a total of 166 coupled self-taken and CT specimens collected. Specimens were stored at room temperature for a maximum of 10 months and analyzed using validated the RIATOL qPCR HPV genotyping test, a quantitative polymerase chain reaction (qPCR) high-throughput HPV E6, E7 assay. The average age of the participating women was 32 years. Seventy-three women (87.9%) felt that NASS was easy to use. An overall HPV, high-risk (HR) HPV, and low-risk HPV prevalence was 22.7% (15/66), 18.2% (12/66), and 6.1% (4/66), respectively. The overall HR HPV prevalence was 17.2% (NASS) and 15.5% (CT). The most prevalent HPV type was HPV51; HPV 16 was only detected in 1 woman (CT+NASS) and HPV18 only in 1 woman (CT). The overall measurement agreement between self-taken and CT samples was moderate with a kappa value of 0.576 (P < .001). Lifetime partnered with >2 men were associated with HR HPV positivity (P < .001). There was a strong statistical association between HR HPV positivity and visual inspection with acetic acid- positive (P < .001). The NASS for HPV testing could be seen as an alternative option and might be acceptable to Ethiopian women. The overall HR HPV prevalence was comparable with Sub-Saharan countries in the general population.


Assuntos
Detecção Precoce de Câncer/métodos , Infecções por Papillomavirus/diagnóstico , Manejo de Espécimes/métodos , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal/métodos , Adulto , Idoso , Etiópia/epidemiologia , Feminino , Genótipo , Humanos , Pessoa de Meia-Idade , Infecções por Papillomavirus/epidemiologia , Infecções por Papillomavirus/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade , Adulto Jovem
14.
Pestic Biochem Physiol ; 158: 47-53, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378360

RESUMO

Buprofezin is a chitin synthesis inhibitor that is very effective against Homopteran pests, such as the white-backed planthopper (WBPH), S. furcifera (Horvath). In the present study, resistance selection, cross-resistance and mechanisms of buprofezin resistance were investigated in this planthopper species. However, the mechanism associated with resistance to growth regulator insecticides (IGRs) remains largely unknown. A resistant strain (Bup-R) with a resistance level (22-fold) to buprofezin was developed through continuous selection for 47 generations from a laboratory susceptible strain (Bup-S). The results showed that the Bup-R exhibited no cross-resistance to other tested insecticides. Synergism tests showed that piperonyl butoxide (PBO) (SR = 3.9-fold) and diethyl maleate (DEM) (SR = 1.8-fold) had synergistic effects on buprofezin toxicity in the resistant strain (F47). Enzyme activity results revealed an approximate 5.7-fold difference in cytochrome P450 monooxygenase and a 2-fold difference in glutathione S-transferase (GST) between the resistant and susceptible strains, suggesting that the increased activity of these two enzymes is likely the main detoxification mechanism involved in resistance to buprofezin in this species. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) and GST genes by quantitative real-time PCR results indicated that sixteen P450 and one GST gene were significantly overexpressed in the Bup-R strain, among which thirteen P450 genes and one GST gene were >2-fold higher than in the Bup-S strain. The present study increases our knowledge of the buprofezin resistance mechanism in S. furcifera and provides a useful reference for integrated pest management (IPM) strategies.


Assuntos
Hemípteros/efeitos dos fármacos , Inseticidas/farmacologia , Tiadiazinas/farmacologia , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemípteros/metabolismo , Proteínas de Insetos/genética , Resistência a Inseticidas/genética , Maleatos/metabolismo , Butóxido de Piperonila/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
15.
Pestic Biochem Physiol ; 158: 69-76, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378363

RESUMO

Sex pheromones are crucial for communication between females and males in moths, and pheromone receptors (PRs) play a key role in peripheral coding of sex pheromones. During the last decade, many PR candidates have been identified based on transcriptome sequencing and bioinformatic analysis, but their detailed functions remain mostly unknown. Here, focusing on four PR candidates of Athetis dissimilis (AdisOR1, AdisOR6, AdisOR11 and AdisOR14) identified in a previous study, we first cloned the full-length cDNAs and determined the tissue expression profiles by quantitative real-time PCR (qPCR). The results revealed that expression of three of these genes were male antennae-specific, while AdisOR11 was similar in expression between male and female antennae. Furthermore, the expression level of AdisOR1 was much higher than those of the other three genes. Then, functional analysis was conducted using Xenopus oocyte system. AdisOR1 responded strongly to the sex pheromone component Z9-14:OH and the potential pheromone component Z9,E12-14:OH, suggesting its important role in the sex pheromone perception; AdisOR14 showed specificity for Z9,E12-14:OH; while AdisOR6 and AdisOR11 did not respond to any of the pheromone components and analogs tested. Taken together, this study contributes to elucidate the molecular mechanism of sex pheromone reception and provides potential targets for development of OR based pest control techniques in A. dissimilis.


Assuntos
Proteínas de Insetos/metabolismo , Lepidópteros/metabolismo , Receptores de Feromonas/metabolismo , Animais , Feminino , Proteínas de Insetos/genética , Lepidópteros/genética , Masculino , Feromônios/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Feromonas/genética
16.
Medicine (Baltimore) ; 98(33): e16773, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31415379

RESUMO

Conventional therapy modalities for advanced breast cancer are problematic, whereas checkpoint blockade immunotherapy has been considered as a promising approach. This study aims to determine programmed death-ligand 1 (PD-L1) expression and methylation status of PD-L1 promoter in primary tumor tissue and metastatic foci of patients with stage IV breast cancer.Clinicopathological data and survival rates of 57 breast cancer patients, who were initially staged IV, and operated for intact tumors, were retrospectively analyzed. Immunohistochemical analysis of PD-L1 using 57 primary tumors, 33 paired metastatic lymph nodes, and 14 paired distant metastases was performed. Additionally, the methylation rate of the PD-L1 gene promoter region was determined with real-time polymerase chain reaction (PCR) analysis in 38 samples.Overall PD-L1 expression in primary tumors was 23.1% (12/52). PD-L1 positivity was reduced in lymph nodes by 15.2% (5/33) and in distant metastases by 21.4% (3/14). PD-L1 expression diverged between primary and metastatic foci in a subset of cases (18.2% for lymph node and 33.3% for distant metastasis). In general, the PD-L1 promoter was not methylated, and mean methylation rates were low (min. 0%-max. 21%). We observed no correlation between PD-L1 expression, promoter methylation, and survival.Neither the expression nor the methylation status of PD-L1 in patients, who were presented with stage IV breast cancer and operated for an intact primary tumor, had a statistically significant relation with survival. Discordance in PD-L1 expression between primary tumor and metastasis should be considered during pathological and clinical management of patients who would undergo checkpoint blockade therapy.


Assuntos
Antígeno B7-H1/metabolismo , Neoplasias da Mama/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/genética , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Neoplasias da Mama/cirurgia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Receptor de Morte Celular Programada 1/genética , Reação em Cadeia da Polimerase em Tempo Real , Estudos Retrospectivos , Turquia
17.
Gene ; 716: 144024, 2019 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-31390541

RESUMO

The young leaves generally accumulate a certain concentration anthocyanins in the dominant species of the subtropical forest, and the changes of anthocyanin synthesis-related enzyme genes expression levels had an important effect on the study photoprotection of anthocyanins in the young leaves of subtropical forests. The determination of anthocyanin synthesis-related enzyme gene sequences and the selection of appropriate reference genes provide a basis for analyzing the functional properties of anthocyanins. In this study, four dominant subtropical forest species (i.e., Schima superba, Castanopsis fissa, Acmena acuminatissima, Cryptocarya concinna) were taken as materials. To obtain the correct nucleotide sequences of anthocyanin-related enzymes, the nucleotide sequences of CHS, DFR and ANS in each dominant species were obtained by sequencing and comparison. Then, to select the most stable reference genes for leaves at different developmental stages and different light conditions, the expression levels of six reference genes, including 18S, Actin, GAPDH, TUB, EF1 and UBQ, were studied by real-time fluorescent quantitative PCR (qRT-PCR), and reference gene stability was analyzed by GeNorm and NormFinder software. The results showed that the expression level of Actin was the most stable in S. superba, A. acuminatissima and C. concinna, and the expression level of GAPDH was the most stable in C. fissa. Finally, the expression levels of the anthocyanin synthesis genes CHS, DFR and ANS were analyzed and found to be consistent with the accumulation trend of anthocyanins in leaves. This study has important theoretical and practical significance for future research into the expression of anthocyanin synthesis-related enzyme genes in the dominant tree species in subtropical forests and reveals that anthocyanin has a photoprotective effect for young leaves in high-light environments.


Assuntos
Antocianinas/biossíntese , Árvores/genética , Aciltransferases/genética , Aciltransferases/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Antocianinas/metabolismo , Florestas , Genes de Plantas , Folhas de Planta/genética , Folhas de Planta/metabolismo , RNA de Plantas/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Alinhamento de Sequência , Análise de Sequência , Árvores/enzimologia , Árvores/metabolismo
18.
Acta Cir Bras ; 34(6): e201900602, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31432993

RESUMO

PURPOSE: To investigate the role and related mechanisms of miR-106a in sepsis-induced AKI. METHODS: Serum from sepsis and healthy patients was collected, sepsis mouse model was established by cecal ligation and puncture (CLP). TCMK-1 cells were treated with lipopolysaccharide (LPS) and transfected with THBS2-small interfering RNA (siTHBS2), miR-106a inhibitor, miR-106a mimics and their negative controls (NCs). The expression of miR-106a, thrombospondin 2 (THBS2), Bax, cleaved caspase-3 and Bcl-2, cell viability, relative caspase-3 activity and TNF-α, IL-1ß, IL-6 content were respectively detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, Cell Counting Kit-8 (CCK-8) and enzyme linked immunosorbent assay (ELISA). The relationship between miR-106a and THBS2 was confirmed by dual luciferase reporter assay. RESULTS: MiR-106a was up-regulated in serum of sepsis patients, CLP-induced mice models and LPS-induced TCMK-1 cells. LPS reduced cell viability and Bcl-2 expression, and increased caspase-3 activity, Bax expression, the content of TNF-α, IL-1ß, IL-6. THBS2 was a target of miR-106a. The decreases of caspase-3 activity, TNF-α, IL-1ß, IL-6, Bax expression and the increases of cell viability, Bcl-2 expression caused by miR-106a knockdown were reversed when THBS2 silencing in LPS-stimulated TCMK-1 cells. CONCLUSION: MiR-106a aggravated LPS-induced inflammation and apoptosis of TCMK-1 cells via regulating THBS2 expression.


Assuntos
Lesão Renal Aguda/metabolismo , Células Epiteliais/patologia , Rim/citologia , MicroRNAs/metabolismo , Sepse/patologia , Trombospondinas/farmacologia , Lesão Renal Aguda/patologia , Adulto , Animais , Apoptose , Estudos de Casos e Controles , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Sepse/metabolismo , Transfecção
19.
Rev Soc Bras Med Trop ; 52: e20180249, 2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31365620

RESUMO

INTRODUCTION: Prevalence of influenza A virus (Flu-A), respiratory syncytial virus (RSV), and human metapneumovirus (hMPV) was assessed in children with acute respiratory infections (ARIs). METHODS: Nasopharyngeal aspirates and throat swabs were subjected to real-time polymerase chain reaction (PCR) to detect RSV and Flu-A and to conventional PCR to detect hMPV. RESULTS: Of the 156 children assessed, 93 (59.6%) carried at least one virus, with 35.9% positive for RSV, 14.1% for hMPV, and 9.6% for Flu-A. The prevalence of co-infections was 2.6%. CONCLUSIONS: The high detection rate may reflect increased sensitivity of real-time PCR compared to traditional PCR and viral culture.


Assuntos
Influenza Humana/epidemiologia , Infecções por Paramyxoviridae/epidemiologia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções Respiratórias/virologia , Adolescente , Criança , Pré-Escolar , Estudos Transversais , Feminino , Humanos , Vírus da Influenza A/genética , Irã (Geográfico)/epidemiologia , Masculino , Metapneumovirus/genética , Nasofaringe/virologia , Reação em Cadeia da Polimerase em Tempo Real , Vírus Sincicial Respiratório Humano/genética , Infecções Respiratórias/epidemiologia
20.
Microbiol Res ; 227: 126296, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421712

RESUMO

Heat shock proteins (Hsp) are important factors in the response of organisms to oscillations in environmental conditions. Although Hsp have been studied for a long time, little is known about this protein class in Trichoderma species. Here we studied the expression of Hsp genes during T. asperellum growth, and mycoparasitism against two phytopathogens: Sclerotinia sclerotiorum and Fusarium oxysporum, as well as during thermal stress. The expression levels of these genes were observed by real-time PCR and they showed to be differentially expressed under these conditions. We verified that the TaHsp26c, TaHsp70b and TaHsp70c genes were differentially expressed over time, indicating that these genes can be developmentally regulated in T. asperellum. Except for TaHsp26a, all other genes analyzed were induced in the post-contact condition when T. asperellum was cultured in a confrontation plate assay against itself. Additionally, TaHsp26b, TaHsp26c, TaHsp90, TaHsp104a and TaHsp104b were induced during initial contact between T. asperellum hyphae, suggesting that these proteins must play a role in the organism´s self-recognition mechanism. When we examined gene expression during mycoparasitism, we observed that some genes were induced both by S. sclerotiorum and F. oxysporum, while others were not induced during interaction with either of the phytopathogens. Furthermore, we observed some genes induced only during confrontation against S. sclerotiorum, indicating that the expression of Hsp genes during mycoparasitism seems to be modulated by the phytopathogen. To assess whether such genes are expressed during temperature oscillations, we analyzed their transcription levels during thermal and cold shock. We observed that except for the TaHsp70c gene, all others presented high transcript levels when T. asperellum was submitted to high temperature (38 °C), indicating their importance in the response to heat stress. The TaHsp70c gene was significantly induced only in cold shock at 4 °C. Our results show the importance of Hsp proteins during self-recognition, mycoparasitism and thermal stress in T. asperellum.


Assuntos
Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/fisiologia , Trichoderma/genética , Sequência de Aminoácidos , Ascomicetos/genética , Fusarium/genética , Resposta ao Choque Térmico/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Interações Microbianas , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de Sequência , Estresse Fisiológico/genética , Temperatura Ambiente , Transcriptoma , Trichoderma/crescimento & desenvolvimento
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