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Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7-methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.
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Camellia sinensis , Camellia sinensis/genética , Camellia sinensis/metabolismo , Teobromina/metabolismo , Cafeína/metabolismo , Hojas de la Planta/metabolismo , Té/metabolismo , Factores de Transcripción/genética , ARN Mensajero/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
BACKGROUND: The early prediction of cerebral palsy (CP) could enable the follow-up of high-risk infants during the neuroplasticity period. This study aimed to explore the predictive value of fidgety movement assessment (FMA) and brain magnetic resonance imaging (MRI) for the development of CP in clinic rehabilitation setting. METHODS: This retrospective observational study included infants who underwent FMA and brain MRI at age nine to 20 weeks at Children's Hospital, Zhejiang University School of Medicine, between March 2018 and September 2019. The area under the receiver operating characteristic curve (AUC), sensitivity, specificity, and accuracy of FMA and MRI for predicting the development of CP were assessed. RESULTS: A total of 258 infants (169 males, gestational age 37.4 ± 3.0 weeks, birth weight 2987.9 ± 757.1 g) were included. Fifteen children had CP after age two years. The diagnostic value of FMA and brain MRI combination showed 86.7% sensitivity (95% confidence interval [CI]: 58.4% to 97.7%), 98.4% specificity (95% CI: 95.6% to 99.5%), and 97.7% accuracy (95% CI: 95.0% to 99.1%); the combination diagnostic value also showed a significantly higher AUC for predicting CP after age two years than FMA alone (AUC: 0.981 vs 0.893, P = 0.013). CONCLUSIONS: The diagnostic value of FMA and brain MRI combination during infancy showed a high predictive value for CP development in clinical rehabilitation setting.
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Parálisis Cerebral , Humanos , Lactante , Masculino , Peso al Nacer , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Parálisis Cerebral/diagnóstico por imagen , Parálisis Cerebral/patología , Imagen por Resonancia Magnética , Movimiento , FemeninoRESUMEN
Identification of the phosphatidylserine (PS) discrepancies occurring on the cellular membrane during apoptotic processes is of the utmost importance. However, monitoring the quantity of PS molecules in real-time at a single-cell level currently remains a challenging task. Here, we demonstrate this objective by leveraging the specific binding and reversible interaction exhibited by the zinc(II) dipyridinamine complex (ZnDPA) with PS. Lipoic acid-functionalized ZnDPA (LP-ZnDPA) was subsequently immobilized onto the surface of an atomic force microscopy cantilever to form a force probe, ALP-ZnDPA, enabling a PS-specific dynamic imaging and detection mode. By utilizing this technique, we can not only create a heat map of the expression level of PS with submicron resolution but also quantify the number of molecules present on a single cell's surface with a detection limit of 1.86 × 104 molecules. The feasibility of the proposed method is demonstrated through the analysis of PS expression levels in different cancer cell lines and at various stages of paclitaxel-induced apoptosis. This study represents the first application of a force probe to quantify PS molecules on the surface of individual cells, providing insight into dynamic changes in PS content during apoptosis at the molecular level and introducing a novel dimension to current detection methodologies.
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Fosfatidilserinas , Imagen Individual de Molécula , Fosfatidilserinas/química , Apoptosis , Membrana Celular/metabolismo , Microscopía de Fuerza Atómica/métodos , Análisis EspectralRESUMEN
This study aimed to determine patient and therapeutic characteristics of patients in the medical intensive care unit (MICU) that contribute to inconsistent results of delirium assessments performed during routine clinical practice. Therefore, electronic health records were reviewed and compared with secondary data collected from the same medical ICU patients who were assessed using the Confusion Assessment Method in the ICU (CAM-ICU). Of 5,241 cases involving 762 patients, 827 (15.78%) cases showed disagreement between assessments. Continuous renal replacement therapy, physical restraint use, and altered mental status were factors that increased the likelihood of inconsistencies between assessments. A significant positive correlation was found between the CAM-ICU disagreement rate and the total number of assessments per month. To maximize the reliability of delirium assessments, individual-targeted approaches considering the patient's level of consciousness and type of treatment implemented are required, along with ensuring a stable, and regulated working environment and customized educational programs.
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Delirio , Humanos , Delirio/diagnóstico , Reproducibilidad de los Resultados , Unidades de Cuidados IntensivosRESUMEN
Pearl of Csaba (PC) is a valuable backbone parent for early-ripening grapevine (Vitis vinifera) breeding, from which many excellent early ripening varieties have been bred. However, the genetic basis of the stable inheritance of its early ripening trait remains largely unknown. Here, the pedigree, consisting of 40 varieties derived from PC, was re-sequenced for an average depth of â¼30×. Combined with the resequencing data of 24 other late-ripening varieties, 5,795,881 high-quality single nucleotide polymorphisms (SNPs) were identified following a strict filtering pipeline. The population genetic analysis showed that these varieties could be distinguished clearly, and the pedigree was characterized by lower nucleotide diversity and stronger linkage disequilibrium than the non-pedigree varieties. The conserved haplotypes (CHs) transmitted in the pedigree were obtained via identity-by-descent analysis. Subsequently, the key genomic segments were identified based on the combination analysis of haplotypes, selective signatures, known ripening-related quantitative trait loci (QTLs), and transcriptomic data. The results demonstrated that varieties with a superior haplotype, H1, significantly (one-way ANOVA, P < 0.001) exhibited early grapevine berry development. Further analyses indicated that H1 encompassed VIT_16s0039g00720 encoding a folate/biopterin transporter protein (VvFBT) with a missense mutation. VvFBT was specifically and highly expressed during grapevine berry development, particularly at veraison. Exogenous folate treatment advanced the veraison of "Kyoho". This work uncovered core haplotypes and genomic segments related to the early ripening trait of PC and provided an important reference for the molecular breeding of early-ripening grapevine varieties.
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Vitis , Vitis/metabolismo , Fitomejoramiento , Perfilación de la Expresión Génica/métodos , Transcriptoma , Frutas/metabolismo , GenómicaRESUMEN
Background and objectives: Coffin-Lowry Syndrome (CLS), a rare neurodegenerative disorder, is mainly diagnosed based on clinical manifestations and molecular analyses. In total, about 20 cases of CLS have been reported in China. Here, we report two cases of CLS in identical twin brothers and examine their potential causative mutations. Methods: The Trio mode was used in this analysis, i.e., DNA from the proband and his parents was sequenced. Furthermore, DNA from the proband's twin brother was used for confirmation. Results: A hemizygous variation was detected in the 11th exon of the RPS6KA3 gene, c.898C>T (p.R300*) of the proband, and the same site variation was detected in his identical twin brother; however, the mutation was not detected in his parents. Conclusions: The RPS6KA3 gene mutation c.898C>T (p.R300*) is the causative factor of familial CLS. The variant detected was reported for the first time in the Chinese population. Additionally, by analyzing the previous literature, we were able to summarize the phenotypic and genetic characteristics of GLS in China.
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Síndrome de Coffin-Lowry , Síndrome de Coffin-Lowry/diagnóstico , Síndrome de Coffin-Lowry/genética , Exones , Humanos , Masculino , Mutación , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , HermanosRESUMEN
Currently, no consensus exists regarding Sotos syndrome in the Chinese population. Here, we present a case of neonatal Sotos syndrome, followed by a retrospective analysis of five cases of neonatal Sotos syndrome, reported in China. The study subject was a twin premature infant, heavier than gestational age, with characteristic facial features, limb shaking, and hypertonia. Transient hypoglycemia, abnormal cranial magnetic resonance imaging, multiple nodules in polycystic kidneys and liver, abnormal hearing, patent ductus arteriosus, and an atrial septal defect were also noted. The subject showed overgrowth and developmental retardation at 3 months of age. Sequencing revealed a novel missense mutation, c.5000C>A, in the nuclear receptor binding the SET domain protein 1 gene, resulting in an alanine-to-glutamate substitution. The bioinformatics analysis suggested high pathogenicity at this site. This study provides insights into diagnosis of neonatal Sotos syndrome based on specific phenotypes. Subsequent treatment and follow-up should focus on developmental retardation, epilepsy, and scoliosis.
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Síndrome de Sotos , Histona Metiltransferasas/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Mutación Missense/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estudios Retrospectivos , Síndrome de Sotos/genéticaRESUMEN
Histone demethylases containing the JmjC domain play an extremely important role in maintaining the homeostasis of histone methylation and are closely related to plant growth and development. Currently, the JmjC domain-containing proteins have been reported in many species; however, they have not been systematically studied in grapes. In this paper, 21 VviJMJ gene family members were identified from the whole grape genome, and the VviJMJ genes were classified into five subfamilies: KDM3, KDM4, KDM5, JMJD6, and JMJ-only based on the phylogenetic relationship and structural features of Arabidopsis and grape. After that, the conserved sites of VviJMJ genes were revealed by protein sequence analysis. In addition, chromosomal localization and gene structure analysis revealed the heterogeneous distribution of VviJMJ genes on grape chromosomes and the structural features of VviJMJ genes, respectively. Analysis of promoter cis-acting elements demonstrated numerous hormone, light, and stress response elements in the promoter region of the VviJMJ genes. Subsequently, the grape fruit was treated with MTA (an H3K4 methylation inhibitor), which significantly resulted in the early ripening of grape fruits. The qRT-PCR analysis showed that VviJMJ genes (except VviJMJ13c) had different expression patterns during grape fruit development. The expression of VviJMJ genes in the treatment group was significantly higher than that in the control group. The results indicate that VviJMJ genes are closely related to grape fruit ripening.
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Arabidopsis , Vitis , Arabidopsis/genética , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/genética , Histonas/metabolismo , Hormonas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vitis/genética , Vitis/metabolismoRESUMEN
ABSTRACT: BACKGROUND: Biosignal data acquired during quantitative electroencephalography (QEEG) research may ultimately be used to develop algorithms for more accurate detection of delirium. This study investigates the biosignal changes during delirium states by using the QEEG data of patients in a medical intensive care unit. METHODS: This observational study was conducted between September 2018 and December 2019 at a tertiary hospital in South Korea. Delirium was measured using the Korean version of Confusion Assessment Method for the Intensive Care Unit in intensive care unit patients. Quantitative EEG measurements were recorded for 20 minutes in a natural state without external treatment or stimuli, and QEEG data measured in the centroparietal and parietal regions with eyes open were selected for analysis. Power spectrum analysis with a 5-minute epoch was conducted on the selected 65 cases. RESULTS: QEEG changes in the presence of delirium indicated that alpha, beta, gamma, and spectral edge frequency 50% waves showed significantly lower absolute power spectra than the corresponding findings in the absence of delirium. Brain-mapping results showed that these brain waves were inactivated in delirious states. CONCLUSION: QEEG assessments can potentially detect the changes in the centroparietal and parietal regions of delirium patients. QEEG changes, including lower power spectra of alpha, beta, and gamma waves, and spectral edge frequency 50%, can be successfully used to distinguish delirium from the absence of delirium.
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Delirio , Algoritmos , Cuidados Críticos , Delirio/diagnóstico , Electroencefalografía/métodos , Humanos , Unidades de Cuidados IntensivosRESUMEN
ABSTRACT: The Griffiths Mental Development Scale-Chinese (GDS-C) is used in China to assess the development of children from birth to 8âyears of age. Language disorders are a common symptom of autism spectrum disorder (ASD) and global developmental delay (GDD)/intellectual disability (ID). There is a need to identify distinct clinical characteristics in children suspected of having these 2 disorders, mainly presenting as language disorders. Here, we aimed to use the GDS-C to evaluate children presenting with language problems to identify characteristics that distinguish ASD and GDD/ID. Children with language problems were recruited between August 2018 and December 2019. A total of 150 children aged 25 to 95.2âmonths were enrolled (50 in the ASD group, 50 in the GDD/ID group, and 50 in the typical group). Each group was subdivided by age as follows: 24-36âmonths, >36-60âmonths, and >60-96âmonths. Developmental characteristics assessed using the GDS-C were analyzed and compared. Both, children with ASD and GDD/ID presented with a lower developmental level than typical children in all six subscales of the GDS-C. No significant differences were observed in the six subscale scores between the ASD and GDD/ID groups, except for the practical reasoning subscale score in the >36 to 60âmonths subgroups, which was significantly lower in the GDD/ID group than in the ASD group. The developmental imbalance of subscales within the ASD and GDD/ID groups identified troughs in the personal-social, language, and practical reasoning areas in children with ASD and in the language and practical reasoning areas in children with GDD/ID relative to typical children. The GDS-C is a useful, comprehensive tool for the assessment of the developmental state of children with ASD and GDD/ID. Characteristics of practical reasoning subscale help diagnose autism in >36 to 60âmonths old children.
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Trastorno del Espectro Autista/diagnóstico , Discapacidades del Desarrollo/diagnóstico , Discapacidad Intelectual/diagnóstico , Trastornos del Lenguaje/diagnóstico , Pruebas Neuropsicológicas , Trastorno del Espectro Autista/complicaciones , Niño , Desarrollo Infantil , Preescolar , China , Discapacidades del Desarrollo/complicaciones , Diagnóstico Diferencial , Estudios de Factibilidad , Femenino , Humanos , Discapacidad Intelectual/complicaciones , Trastornos del Lenguaje/etiología , Masculino , TraduccionesRESUMEN
In contrast to emotional and behavioral problems (EBPs), which can disrupt normal adolescent development, resilience can buffer the effects of stress and adverse childhood experiences and can help youth overcome adversity. While research has looked at the relationship between adolescent resilience and EBPs, current literature relatively lack a discussion of a strengths-based approach of resilience framework, nor discuss non-western sociocultural contexts. In this study, we utilized the resilience theory to examine the effects of individual mindfulness and life skills on resilience and consequently on EBPs in a group of low-income and gifted adolescents in China. A secondary data of 152 adolescents from a specialized school for low-income and gifted students in Guangzhou, China was used for the analysis. The findings from structural equation modeling indicated that mindfulness and life skills were associated with heightened resilience and reduced EBPs. In addition, resilience reduced EBPs for this group of adolescents. These findings underscore the promise of mindfulness and life skills training on increasing resilience and reducing EBPs in gifted adolescents.
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The Alberta Infant Motor Scale (AIMS) is widely used to screen for delays in motor development in high-risk infants, but its reliability and validity in Chinese infants have not been investigated. To examine the reliability and concurrent validity of AIMS in high-risk infants aged 0-9 months in China, this single-center study enrolled 50 high-risk infants aged 0-9 months (range, 0.17-9.27; average, 4.14±2.02), who were divided into two groups: 0-3 months (n=23) and 4-9 months (n=27). A physical therapist evaluated the infants with AIMS, with each evaluation video-recorded. To examine interrater reliability, two other evaluators calculated AIMS scores by observing the videos. To measure intrarater reliability, the two evaluators rescored AIMS after >1 month, using the videos. Concurrent validity was assessed by comparing results between AIMS and the Peabody Developmental Motor Scale-2 (PDMS-2). For all age groups analyzed (0-3, 4-9, and 0-9 months), intraclass correlation coefficients (ICCs) for AIMS total score were high for both intrarater comparisons (0.811-0.995) and interrater comparisons (0.982-0.997). AIMS total scores were well correlated with all PDMS-2 subtest scores (ICC=0.751-0.977 for reflexes, stationary, locomotion, grasping, and visual-motor integration subsets). However, the fifth percentile of AIMS total score was only moderately correlated with the gross motor quotient, fine motor quotient, and total motor quotient subtests of PDMS-2 (kappa=0.580, 0.601, and 0.724, respectively). AIMS has acceptable reliability and concurrent validity for screening of motor developmental delay in high-risk infants in China.
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Desarrollo Infantil , Destreza Motora , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
Infantile neuroaxonal dystrophy (INAD) is a rare neurodegenerative disease with early onset. PLA2G6 gene mutations have been identified in the majority individuals with INAD. In future, molecular diagnosis of INAD will replace the invasive biopsies used previously. In the present report, monozygotic male twins with INAD were referred The Children's Hospital (Zhejiang University School of Medicine, Zhejiang, China) at fifteen months old for delayed development. The older brother was found to have developmental stagnation when he was 6 months old. The patient could not stand securely without support, and had poor eye tracking and listening ability. Magnetic resonance imaging (MRI) of the patient's brain revealed cerebellar atrophy and electromyography identified signs of peripheral neuropathy. The younger brother displayed similar clinical features and findings. Two different phospholipase A2 group VI (PLA2G6; 22q13.1) gene mutations were detected in the twins by DNA sequencing. The results of the present study indicate that neurogenetic disease should be considered when child patients present with idiopathic developmental stagnation, particularly when similar cases have appeared in the same family. In addition, INAD should be considered as a possible diagnosis when the patient has developmental delay of the central and peripheral nerves. In the future, molecular genetic testing will be the primary method of INAD diagnosis, enabling better prevention of this genetic disease.
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BACKGROUND: Claudin-6 is a candidate tumor suppressor gene in breast cancer, and has been shown to be regulated by DNA methylation and histone modification in breast cancer lines. However, the expression of claudin-6 in breast invasive ductal carcinomas and correlation with clinical behavior or expression of other markers is unclear. We considered that the expression pattern of claudin-6 might be related to the expression of DNA methylation associated proteins (methyl-CpG binding protein 2 (MeCP2) and DNA methyltransferase 1 (DNMT1)) and histone modification associated proteins (histone deacetylase 1 (HDAC1), acetyl-histone H3 (H3Ac) and acetyl- histone H4 (H4Ac)). METHODS: We have investigated the expression of claudin-6, MeCP2, HDAC1, H3Ac and H4Ac in 100 breast invasive ductal carcinoma tissues and 22 mammary gland fibroadenoma tissues using immunohistochemistry. RESULTS: Claudin-6 protein expression was reduced in breast invasive ductal carcinomas (P < 0.001). In contrast, expression of MeCP2 (P < 0.001), DNMT1 (P = 0.001), HDAC1 (P < 0.001) and H3Ac (P = 0.004) expressions was increased. Claudin-6 expression was inversely correlated with lymph node metastasis (P = 0.021). Increased expression of HDAC1 was correlated with histological grade (P < 0.001), age (P = 0.004), clinical stage (P = 0.007) and lymph node metastasis (P = 0.001). H3Ac expression was associated with tumor size (P = 0.044) and clinical stage of cancers (P = 0.034). MeCP2, DNMT1 and H4Ac expression levels did not correlate with any of the tested clinicopathological parameters (P > 0.05). We identified a positive correlation between MeCP2 protein expression and H3Ac and H4Ac protein expression. CONCLUSIONS: Our results show that claudin-6 protein is significantly down-regulated in breast invasive ductal carcinomas and is an important correlate with lymphatic metastasis, but claudin-6 down-regulation was not correlated with upregulation of the methylation associated proteins (MeCP2, DNMT1) or histone modification associated proteins (HDAC1, H3Ac, H4Ac). Interestingly, the expression of MeCP2 was positively correlated with the expression of H3Ac and H3Ac protein expression was positively correlated with the expression of H4Ac in breast invasive ductal carcinoma VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4549669866581452.
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Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Adulto , Anciano , Anciano de 80 o más Años , Claudinas/análisis , Claudinas/biosíntesis , Femenino , Histona Desacetilasa 1/análisis , Histona Desacetilasa 1/biosíntesis , Histonas/análisis , Histonas/biosíntesis , Humanos , Inmunohistoquímica , Proteína 2 de Unión a Metil-CpG/análisis , Proteína 2 de Unión a Metil-CpG/biosíntesis , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Proteínas Represoras/análisis , Proteínas Represoras/biosíntesisRESUMEN
AIM: To establish the rapid, specific, and sensitive method for detecting O157:H7 with DNA microchips. METHODS: Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and virulent genes of E. coli O157:H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-labeled single prime. Hybridization was performed for 60 min at 45 degrees. Microchip images were taken using a confocal fluorescent scanner. RESULTS: Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157:H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157:H7 fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-labeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization. CONCLUSION: Microarray analysis of O157:H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.
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Infecciones por Escherichia coli/diagnóstico , Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Técnicas de Diagnóstico Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/genética , Colorantes Fluorescentes/metabolismo , Humanos , Sensibilidad y EspecificidadRESUMEN
A DNA array for rapid detection and genotyping of the pathogenic microbes of epidemic hemorrhagic fever, tsutsugamushi disease, leptospirosis, malaria, schistosomiasis, cholera, and hemorrhagic colitis was developed. The specific and relatively conserved PCR primers and DNA probes were screened from the characteristic genes of the pathogenic microbes. The PCR or RT-PCR methods were established for amplifying and labeling the DNA fragments of the pathogenic microbes. All the probes with the same Tm value were synthesized chemically and modified with an NH2 at their 5' terminus, they were printed on glass slides for fabrication of a oligonucleotide DNA array. The developed DNA array could be used for detecting and genotyping the pathogenic microbes simultaneously, and they had a high sensitivity and specificity.
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Técnicas Microbiológicas , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Parasitología/métodos , Animales , Secuencia de Bases , Cartilla de ADN/genética , Sondas de ADN/genética , Enterobacteriaceae/genética , Enterobacteriaceae/aislamiento & purificación , Genotipo , Virus Hantaan/genética , Virus Hantaan/aislamiento & purificación , Humanos , Leptospira interrogans/genética , Leptospira interrogans/aislamiento & purificación , Nanotecnología , Orientia tsutsugamushi/genética , Orientia tsutsugamushi/aislamiento & purificación , Plasmodium/genética , Plasmodium/aislamiento & purificación , Schistosoma/genética , Schistosoma/aislamiento & purificación , Vibrio cholerae/genética , Vibrio cholerae/aislamiento & purificaciónRESUMEN
AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood. METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected. RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively. CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.
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Variación Genética , Virus de la Hepatitis B/genética , Hepatitis B/virología , Secuencia de Bases , China , Cartilla de ADN , ADN Viral/sangre , ADN Viral/genética , Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Humanos , Mutación , Reacción en Cadena de la Polimerasa/métodosRESUMEN
AIM: The hepatitis B surface antigen (HBsAg) is considered to be one of the best markers for the diagnosis of acute and chronic HBV infection. But in some patients, this antigen cannot be detected by routine serological assays despite the presence of virus. One of the most important explanations for the lack of detectable HBsAg is that mutations which occur within the "a" determinant of HBV S gene can alter expression of HBsAg and lead to changes of antigenicity and immunogenicity of HBsAg accordingly. As a result, these mutants cannot be detected by diagnosis assays. Thus, it is essential to find out specific and sensitive methods to test the new mutants and further investigate their distribution. This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551. METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS, P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3' terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, we explored the specific conditions for amplifying HBV DNAs with a mutation at nt551 by regulating annealing temperature and the concentration of these primers. 126 serum samples from patients of hepatitis B were collected, among which 16 were positive for HBV S DNA in the nested PCR amplification. These 16 HBV S DNAs were detected by using the msPCR method. RESULTS: When the annealing temperature was raised to 71 degrees, nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS; At 72 degrees and 5 pmole of the primers (each) in reaction of 25 microl volume, nt551C and nt551T were amplified specifically by P551C-PPS and P551T-PPS. 16 of HBV S gene fragments were characterized by using this method. 14 of them were positive for nt551A, one was positive for nt551G, and the other one was positive for nt551T. The results were confirmed by nucleotide sequencing. CONCLUSION: The mutation specific polymerase chain reaction is a specific and sensitive method for detecting the mutations of HBV genome at nt551.