RESUMEN
The exceptional longevity of Ames dwarf (DF) mice can be abrogated by a brief course of growth hormone (GH) injections started at 2 weeks of age. This transient GH exposure also prevents the increase in cellular stress resistance and decline in hypothalamic inflammation characteristic of DF mice. Here, we show that transient early-life GH treatment leads to permanent alteration of pertinent changes in adipocytes, fat-associated macrophages, liver, muscle, and brain that are seen in DF mice. Ames DF mice, like Snell dwarf and GHRKO mice, show elevation of glycosylphosphatidylinositol specific phospholipase D1 in liver, neurogenesis in brain as indicated by BDNF and DCX proteins, muscle production of fibronectin type III domain-containing protein 5 (a precursor of irisin), uncoupling protein 1 as an index of thermogenic capacity in brown and white fat, and increase in fat-associated anti-inflammatory macrophages. In each case, transient exposure to GH early in life reverts the DF mice to the levels of each protein seen in littermate control animals, in animals evaluated at 15-18 months of age. Thus, many of the traits seen in long-lived mutant mice, pertinent to age-related changes in inflammation, neurogenesis, and metabolic control, are permanently set by early-life GH levels.
Asunto(s)
Hormona del Crecimiento , Hormona de Crecimiento Humana , Adipocitos/metabolismo , Animales , Encéfalo/metabolismo , Hormona del Crecimiento/metabolismo , Hormona de Crecimiento Humana/metabolismo , Inflamación/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Mutantes , Músculos/metabolismoRESUMEN
Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as the gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative real-time PCR (OSN-qRT-PCR) assay for the highly sensitive detection of SARS-CoV-2 targeting the ORF1ab and N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of the commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results and 7 had no cycle threshold (CT) values) were detected by OSN-qRT-PCR. Moreover, the 7 qRT-PCR-positives in the qRT-PCR gray zone (CT values of ORF1ab ranged from 37.48 to 39.07, and CT values of N ranged from 37.34 to 38.75) were out of the gray zone and thus were deemed to be positive by OSN-qRT-PCR, indicating that the positivity of these samples is confirmative. Compared to the qRT-PCR kit, the OSN-qRT-PCR assay revealed higher sensitivity and specificity, showing better suitability to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.
Asunto(s)
Infecciones por Coronavirus/diagnóstico , Neumonía Viral/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto , Anciano , Anciano de 80 o más Años , Betacoronavirus/genética , Betacoronavirus/aislamiento & purificación , COVID-19 , Infecciones por Coronavirus/virología , Proteínas de la Nucleocápside de Coronavirus , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de la Nucleocápside/genética , Pandemias , Fosfoproteínas , Neumonía Viral/virología , Poliproteínas , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2 , Sensibilidad y Especificidad , Proteínas Virales/genéticaRESUMEN
Hepatitis B virus (HBV) is a widespread blood-borne pathogen associated with the complication of liver cirrhosis and hepatocellular carcinoma, particularly in south-east Asian and African countries where HBV is highly endemic and the budget and resources are limited. Therefore, simple, rapid, and portable field detection methods are crucial to efficiently control HBV infection. In this study, using heat-treated DNA, we developed two-field applicable detection assays for HBV based on recombinase-aided amplification (RAA). One was an internal controlled duplex RAA assay using a portable real-time fluorescence detection device, another was an instrument-free visual observation assay using lateral flow dipsticks. The entire experimental time was greatly shortened to less than 40 minutes at 39.0°C. The sensitivities, specificities, and clinical performance of both assays were evaluated. Compared with quantitative polymerase chain reaction assay as a reference, our results demonstrated that the two RAA-based assay obtained 97.18% and 95.77% of sensitivity, respectively, and the specificity was 100%, by testing a total of 157 serum samples with HBsAg positive. We conclude that the advantages of rapidity, simplicity, portability, and visualization of proposed two assays make them great potentials in point-of-care testing of HBV infection by untrained people in resource-limited situations.
RESUMEN
Cervical cancer is primarily caused by persistent infection with high-risk human papillomavirus (HPV), and 70% of cases are associated with HPV16 and 18 infections. The objective of this study was to establish rapid, simple, and sensitive internally controlled recombinase-aided amplification (IC-RAA) assays for the detection of HPV16 and 18. The assays were performed at 39 â and were completed within 30 min. A total of 277 clinical samples of exfoliated cervical cells were tested by IC-RAA assays and commercial HPV real-time fluorescent PCR kits using extracted DNA and samples treated with nucleic acid releasing agent. The analytical sensitivity of the IC-RAA assay was found to be 10 copies/µL for the detection of HPV16 and 18 when using recombinant plasmids as targets. The optimal concentration of the internal control (IC) plasmid and 18 was 1000 copies/µL for HPV16 and 100 copies/µL for HPV18. The clinical sensitivity of the IC-RAA assays for HPV16 using extracted DNA and samples treated with nucleic acid releasing agent was 98.73% and 97.47%, respectively, with kappa values of 0.977 (P < 0.01) and 0.955 (P < 0.01), respectively, and 100% The specificity in both cases. For HPV18, the sensitivity and specificity were 100%, and the kappa value was 1 for both samples (P < 0.01). The IC-RAA assay is a promising tool for the detection of HPV16 and HPV18, especially in resource-constrained settings.
Asunto(s)
ADN Viral/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Técnicas de Amplificación de Ácido Nucleico , Infecciones por Papillomavirus/diagnóstico , Neoplasias del Cuello Uterino/diagnóstico , Adolescente , Adulto , Anciano , Cuello del Útero/patología , Cuello del Útero/virología , Cartilla de ADN/síntesis química , Cartilla de ADN/genética , Células Epiteliales/patología , Células Epiteliales/virología , Femenino , Papillomavirus Humano 16/clasificación , Papillomavirus Humano 16/aislamiento & purificación , Papillomavirus Humano 18/clasificación , Papillomavirus Humano 18/aislamiento & purificación , Humanos , Persona de Mediana Edad , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Juego de Reactivos para Diagnóstico , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
To investigate the effect of miR-223 on thyroid cancer cells, further to study its potential mechanisms. The difference in miR-223 expression between normal thyroid Nthy-ori3-l cells and thyroid cancer SW579 cells was detected by PCR. The miR-223 overexpression and silencing vector transfection were verified by qRT-PCR. To further investigate the role of miR-223 in AQP-1, the AQP-1 siRNA vector was transfected on the basis of transfection of miR-223 inhibitor vector. The cell proliferation was detected by plate cloning, MTT, and cellular immunofluorescence assays. Cell cycle and apoptosis were detected by flow cytometry. Western blot was used to detect the expression of AQP-1 protein. The expression of miR-223 in SW579 cells was higher than that in normal cells. After transfection with miR-223 mimic, miR-223 expression was increased in SW579 cells. MiR-223 inhibitor transfection can inhibit SW579 cells proliferation, promote apoptosis, and inhibit cell cycle G0/G1 arrest. The SW579 cells proliferation was decreased, and the apoptosis rate was increased after transfection of AQP-1 silencing vector. Compared with the AQP-1 siRNA group, the SW579 cells proliferation rate was further reduced, and the apoptosis rate was significantly increased after co-transfection of miR-223 silencing vector and AQP-1 silencing vector. AQP-1 protein was highly expressed in SW579 cells, and miR-223 inhibitor can down-regulate the expression of APQ-1 protein. The expression AQP-1 protein was significantly reduced after transfected with AQP-1 silencing vector. Inhibition of miR-223 expression could suppress proliferation and promote apoptosis of SW579, and its mechanism is related to down-regulation of APQ-1 protein expression.
Asunto(s)
Acuaporina 1/genética , Proliferación Celular/genética , MicroARNs/genética , Neoplasias de la Tiroides/genética , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen , Humanos , MicroARNs/antagonistas & inhibidores , ARN Interferente Pequeño/genética , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Neoplasias de la Tiroides/patologíaRESUMEN
BACKGROUND: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control. METHODS: We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min. RESULTS: The analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol. CONCLUSIONS: We provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.
Asunto(s)
Adenovirus Humanos/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/normas , Recombinasas/genética , Adenovirus Humanos/genética , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Infecciones del Sistema Respiratorio/epidemiología , Sensibilidad y Especificidad , Serogrupo , TemperaturaRESUMEN
BACKGROUND: Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two main etiological agents of Hand, Foot and Mouth Disease (HFMD). Simple and rapid detection of EV71 and CA16 is critical in resource-limited settings. METHODS: Duplex real time reverse-transcription recombinase aided amplification (RT-RAA) assays incorporating competitive internal amplification controls (IAC) and visible RT-RAA assays combined with lateral flow strip (LFS) for detection of EV71 and CA16 were developed respectively. Duplex real time RT-RAA assays were performed at 42 °C within 30 min using a portable real-time fluorescence detector, while LFS RT-RAA assays were performed at 42 °C within 30 min in an incubator. Recombinant plasmids containing conserved VP1 genes were used to analyze the sensitivities of these two methods. A total of 445 clinical specimens from patients who were suspected of being infected with HFMD were used to evaluate the performance of the assays. RESULTS: The limit of detection (LoD) of the duplex real time RT-RAA for EV71 and CA16 was 47 copies and 38 copies per reaction, respectively. The LoD of the LFS RT-RAA for EV71 and CA16 were both 91 copies per reaction. There was no cross reactivity with other enteroviruses. Compared to reverse transcription-quantitative PCR (RT-qPCR), the clinical diagnostic sensitivities of the duplex real time RT-RAA assay were 92.3% for EV71 and 99.0% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. The clinical diagnostic sensitivities of the LFS RT-RAA assay were 90.1% for EV71 and 94.9% for CA16, and the clinical diagnostic specificities were 99.7 and 100%, respectively. CONCLUSIONS: The developed duplex real time RT-RAA and LFS RT-RAA assays for detection of EV71 and CA16 are potentially suitable in primary clinical settings.
Asunto(s)
Enterovirus Humano A/aislamiento & purificación , Enterovirus/aislamiento & purificación , Enfermedad de Boca, Mano y Pie/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Enterovirus/genética , Enterovirus Humano A/genética , Humanos , Sensibilidad y EspecificidadRESUMEN
Human respiratory syncytial virus (RSV) is a common viral pathogen that causes lower respiratory tract infections in infants and children globally. In this study, we developed a duplex reverse transcription recombinase-aided amplification (duplex-rtRAA) assay containing an internal control in a single closed tube for the detection of human RSV. The internal control in the amplification effectively eliminated false-negative results and ensured the accuracy of the duplex-rtRAA system. We first developed and evaluated a universal singleplex-rtRAA assay for RSV. The sensitivity of this assay for RSV was determined as 4.4 copies per reaction, and the specificity was 100%. Next, a duplex-rtRAA assay with an internal control was established. The sensitivity of the duplex-rtRAA assay approached 5.0 copies per reaction, and no cross-reaction with other common respiratory viruses was observed. The two detection methods (singleplex-rtRAA and duplex-rtRAA) developed in this study were used to test 278 clinical specimens, and the results showed absolute consistency with RSV RT-qPCR analysis, demonstrating 100% diagnostic sensitivity and specificity. These data indicate that the duplex-rtRAA has great potential for the rapid detection of RSV with a high sensitivity.
Asunto(s)
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/análisis , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitial Respiratorio Humano/genética , Infecciones del Sistema Respiratorio/diagnóstico , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa , Recombinasas , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Infecciones del Sistema Respiratorio/virología , Transcripción Reversa , Sensibilidad y EspecificidadRESUMEN
Hand, foot and mouth disease (HFMD) is a serious public health problem, and coxsackievirus A6 (CVA6) and coxsackievirus A10 (CVA10) are two of the major causative pathogens, in addition to enterovirus 71 (EV71) and coxsackievirus A16 (CVA16). A simple and rapid reverse transcription recombinase-aided amplification assay (RT-RAA) was developed for the detection of CVA10 and CVA6 in this study. The analytical sensitivity for detection of CVA10 and CVA6 at 95% probability by probit regression analysis was 35 copies per reaction and 38 copies per reaction, respectively, with 100% specificity. Compared with commercial RT-qPCR assays, when testing 455 fecal specimens, the kappa value of the RT-RAA assay for CVA10 and CVA6 was 0.920 (p < 0.001) and 0.952 (p < 0.001), respectively. Moreover, four samples that were positive for CVA10 and five that were positive for CVA6 by RT-RAA but negative by RT-qPCR were further determined to be true positives. These results demonstrate that the proposed RT-RAA assays are very valuable tools for the detection of CVA10 and CVA6 and have potential for use in resource-limited settings.
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Enterovirus/genética , Enfermedad de Boca, Mano y Pie/diagnóstico , ARN Viral/genética , Recombinasas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Niño , Preescolar , Cartilla de ADN/química , Cartilla de ADN/genética , Enterovirus/clasificación , Enterovirus/aislamiento & purificación , Heces/virología , Femenino , Enfermedad de Boca, Mano y Pie/virología , Humanos , Lactante , Masculino , Plásmidos/química , Plásmidos/metabolismo , Recombinasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Sensibilidad y EspecificidadRESUMEN
Respiratory viruses are recognized as serious causes of morbidity and mortality in lower respiratory tract infections in patients. Isothermal amplification assays are increasingly used in their detection because of their rapidity, simplicity and cost-effectiveness, when compared to traditional molecular diagnostic methods. However, systematic assessment of these assays in the clinical settings is rarely reported. MEDLINE (Pubmed) searches were done analysing subject headings and words in the abstract related to isothermal amplification assay and virus. Selected loop-mediated isothermal amplification assays (LAMP) for respiratory syncytial virus (RSV), human metapneumovirus (HMPV) and adenovirus (ADV) as well as a reverse transcription genome exponential amplification reaction assay (GEAR) for human rhinovirus (HRV) were clinically evaluated in a head to head comparison against a two-tube multiplex reverse transcription-PCR (RT-PCR) assay (two-tube assay) using 634 respiratory specimens from children with pneumonia from different regions in China. Discrepant results between isothermal amplification assays and the two-tube assay were resolved by sequencing. A comparison of sensitivities of each selected isothermal amplification assay among province, gender, and age groups was also analyzed. A total of 634 respiratory specimens selected from Hebei Province children's hospital and Hunan Provincial Center for Disease Control and Prevention were tested. The overall detection rate (number of positive specimens/total specimens) for viruses tested by Reverse transcription (RT)-LAMP/LAMP/RT-GEAR was 35.9% while the detection rate was 46.2% by the two-tube assay. The sensitivity of each isothermal amplification assay was 88.4%, 74.3%, 100% and 73.6% for RSV, HMPV, ADV and HRV, respectively. No false positives were found in isothermal amplification assays. All the discrepant negative results by isothermal amplification assays were confirmed false negatives by sequencing. The LAMP assay for ADV showed significant consistency with the two-tube assay. A higher sensitivity of RSV detection was found in Hunan Province than in Hebei Province (P = 0.01). Among different age groups, a higher sensitivity of RSV detection was also found in children older than 1 year, when compared to children less than 1 year (P = 0.01). The clinical performance of the selected isothermal amplification assays for different viruses varies. Multiple-center assessment is critical to evaluate isothermal amplification assays, especially for RNA viruses, for their broad use in clinical hospital. The selected LAMP assay for the detection of ADV is reliable and has great potential for use in clinics; however, the sensitivities of the LAMP/GEAR assays for the detection of RSV, HMPV and HRV need to be further improved to meet clinical requirements, although a statistical difference in sensitivity was only found for the selected LAMP assay for RSV.
Asunto(s)
Adenoviridae/genética , Metapneumovirus/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Neumonía Viral/diagnóstico , Virus Sincitial Respiratorio Humano/genética , Rhinovirus/genética , Infecciones por Adenoviridae/diagnóstico , Infecciones por Adenoviridae/virología , Preescolar , China , Femenino , Humanos , Lactante , Masculino , Reacción en Cadena de la Polimerasa Multiplex , Infecciones por Paramyxoviridae/diagnóstico , Infecciones por Paramyxoviridae/virología , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/virología , Neumonía Viral/virología , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción ReversaRESUMEN
Human rhinoviruses (HRVs) have long been recognized as the cause of more than one-half of acute viral upper respiratory illnesses, and they are associated with more-serious diseases in children, such as asthma, acute otitis media and pneumonia. A rapid and universal test for of HRV infection is in high demand. In this study, a reverse transcription genome exponential amplification reaction (RT-GEAR) assay targeting the HRV 5' untranslated region (UTR) was developed for pan-HRV detection. The reaction was performed in a single tube in one step at 65 °C for 60 min using a real-time fluorometer (Genie(®)II; Optigene). The RT-GEAR assay showed no cross-reactivity with common human enteroviruses, including HEV71, CVA16, CVA6, CVA10, CVA24, CVB5, Echo30, and PV1-3 or with other common respiratory viruses including FluA H3, FluB, PIV1-4, ADV3, RSVA, RSVB and HMPV. With in vitro-transcribed RNA containing the amplified regions of HRV-A60, HRV-B06 and HRV-C07 as templates, the sensitivity of the RT-GEAR assay was 5, 50 and 5 copies/reaction, respectively. Experiments to evaluate the clinical performance of the RT-GEAR assay were also carried out with a panel of 143 previously verified samples, and the results were compared with those obtained using a published semi-nested PCR assay followed by sequencing. The tested panel comprised 91 HRV-negative samples and 52 HRV-positive samples (18 HRV-A-positive samples, 3 HRV-B-positive samples and 31 HRV-C-positive samples). The sensitivity and specificity of the pan-HRVs RT-GEAR assay was 98.08 % and 100 %, respectively. The kappa correlation between the two methods was 0.985. The RT-GEAR assay based on a portable Genie(®)II fluorometer is a sensitive, specific and rapid assay for the universal detection of HRV infection.
Asunto(s)
Infecciones por Picornaviridae/diagnóstico , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rhinovirus/genética , Genoma Viral , Humanos , Infecciones por Picornaviridae/virología , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Transcripción Reversa , Rhinovirus/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
The integrative Vaccine Investigation and Online Information Network (VIOLIN) vaccine research database and analysis system (http://www.violinet.org) curates, stores, analyses and integrates various vaccine-associated research data. Since its first publication in NAR in 2008, significant updates have been made. Starting from 211 vaccines annotated at the end of 2007, VIOLIN now includes over 3240 vaccines for 192 infectious diseases and eight noninfectious diseases (e.g. cancers and allergies). Under the umbrella of VIOLIN, >10 relatively independent programs are developed. For example, Protegen stores over 800 protective antigens experimentally proven valid for vaccine development. VirmugenDB annotated over 200 'virmugens', a term coined by us to represent those virulence factor genes that can be mutated to generate successful live attenuated vaccines. Specific patterns were identified from the genes collected in Protegen and VirmugenDB. VIOLIN also includes Vaxign, the first web-based vaccine candidate prediction program based on reverse vaccinology. VIOLIN collects and analyzes different vaccine components including vaccine adjuvants (Vaxjo) and DNA vaccine plasmids (DNAVaxDB). VIOLIN includes licensed human vaccines (Huvax) and veterinary vaccines (Vevax). The Vaccine Ontology is applied to standardize and integrate various data in VIOLIN. VIOLIN also hosts the Ontology of Vaccine Adverse Events (OVAE) that logically represents adverse events associated with licensed human vaccines.
Asunto(s)
Bases de Datos Genéticas , Vacunas/inmunología , Adyuvantes Inmunológicos , Antígenos/química , Antígenos/genética , Minería de Datos , Genes , Genómica , Humanos , Internet , Plásmidos/genética , Proteínas/inmunología , Alineación de Secuencia , Programas Informáticos , Integración de Sistemas , Vacunas/efectos adversos , Vacunas/química , Vacunas/genética , Vacunas Atenuadas/genética , Vacunas de ADN/genética , Factores de Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
Over the past 10 years, genomes of cultivated rice cultivars and their wild counterparts have been sequenced although most efforts are focused on genome assembly and annotation of two major cultivated rice (Oryza sativa L.) subspecies, 93-11 (indica) and Nipponbare (japonica). To integrate information from genome assemblies and annotations for better analysis and application, we now introduce a comparative rice genome database, the Rice Genome Knowledgebase (RGKbase, http://rgkbase.big.ac.cn/RGKbase/). RGKbase is built to have three major components: (i) integrated data curation for rice genomics and molecular biology, which includes genome sequence assemblies, transcriptomic and epigenomic data, genetic variations, quantitative trait loci (QTLs) and the relevant literature; (ii) User-friendly viewers, such as Gbrowse, GeneBrowse and Circos, for genome annotations and evolutionary dynamics and (iii) Bioinformatic tools for compositional and synteny analyses, gene family classifications, gene ontology terms and pathways and gene co-expression networks. RGKbase current includes data from five rice cultivars and species: Nipponbare (japonica), 93-11 (indica), PA64s (indica), the African rice (Oryza glaberrima) and a wild rice species (Oryza brachyantha). We are also constantly introducing new datasets from variety of public efforts, such as two recent releases-sequence data from â¼1000 rice varieties, which are mapped into the reference genome, yielding ample high-quality single-nucleotide polymorphisms and insertions-deletions.
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Bases de Datos Genéticas , Evolución Molecular , Genoma de Planta , Anotación de Secuencia Molecular , Oryza/genética , Genómica , Internet , Proteínas de Plantas/genética , Polimorfismo Genético , Biosíntesis de Proteínas , Secuencias Repetitivas de Ácidos Nucleicos , Programas Informáticos , Transcripción Genética , Interfaz Usuario-ComputadorRESUMEN
OBJECTIVE: To develop a simple, rapid and sensitive colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of coxsackievirus A6 (CV-A6) based on the colour chang of hydroxy naphthol blue (HNB). METHODS: The method employed a set of six primers that recognized sequences of VP1 gene for amplification of nucleic acid under isothermal conditions at 63 °C for 50 min. The products were detected through visual inspection of color change by the pre-addition of HNB dye. The specificity was validated by detecting a collection of different human enteroviruses. The sensitivity of this assay was evaluated by serial dilutions of RNA molecules from in vitro transcription of CV-A6 VP1 gene, and compared with real-time RT-PCR (rRT-PCR) in parallel. This assay was evaluated with 92 clinical specimens from patients with hand-foot-mouth disease. RESULTS: A positive color (sky blue) was only observed in the preparation of CV-A6, whereas none of the other 23 kinds of human enteroviruses showed a color change. The HNB based RT-LAMP showed a sensitivity of 100 copies/reaction, which was at the same level as that of the rRT-PCR. The result of RT-LAMP in analysis of 92 clinical specimens was consistent with that of the rRT-PCR. The kappa correlation between the two methods was 1 and both of the sensitivity and specificity of the RT-LAMP assay were 100%. CONCLUSION: The established RT-LAMP assay had good specificity and sensitivity and thus demonstrated to be a promising screening tool for CV-A6 infection. It also has the potential to be used in resource-limited clinical sites and field study.
Asunto(s)
Colorimetría , Enterovirus/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Colorantes/química , Cartilla de ADN , Enfermedad de Boca, Mano y Pie/virología , Humanos , Indicadores y Reactivos/química , Naftalenosulfonatos/química , Reacción en Cadena en Tiempo Real de la Polimerasa , Transcripción Reversa , Sensibilidad y EspecificidadRESUMEN
Since the first DNA vaccine studies were done in the 1990s, thousands more studies have followed. Here we report the development and analysis of DNAVaxDB (http://www.violinet.org/dnavaxdb), the first publically available web-based DNA vaccine database that curates, stores, and analyzes experimentally verified DNA vaccines, DNA vaccine plasmid vectors, and protective antigens used in DNA vaccines. All data in DNAVaxDB are annotated from reliable resources, particularly peer-reviewed articles. Among over 140 DNA vaccine plasmids, some plasmids were more frequently used in one type of pathogen than others; for example, pCMVi-UB for G- bacterial DNA vaccines, and pCAGGS for viral DNA vaccines. Presently, over 400 DNA vaccines containing over 370 protective antigens from over 90 infectious and non-infectious diseases have been curated in DNAVaxDB. While extracellular and bacterial cell surface proteins and adhesin proteins were frequently used for DNA vaccine development, the majority of protective antigens used in Chlamydophila DNA vaccines are localized to the inner portion of the cell. The DNA vaccine priming, other vaccine boosting vaccination regimen has been widely used to induce protection against infection of different pathogens such as HIV. Parasitic and cancer DNA vaccines were also systematically analyzed. User-friendly web query and visualization interfaces are available in DNAVaxDB for interactive data search. To support data exchange, the information of DNA vaccines, plasmids, and protective antigens is stored in the Vaccine Ontology (VO). DNAVaxDB is targeted to become a timely and vital source of DNA vaccines and related data and facilitate advanced DNA vaccine research and development.
Asunto(s)
Bases de Datos de Ácidos Nucleicos , Vacunas de ADN/inmunología , Antígenos Bacterianos/inmunología , Bacterias/inmunología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/prevención & control , Vacunas Bacterianas/inmunología , Vacunas contra el Cáncer/inmunología , Bases de Datos de Ácidos Nucleicos/estadística & datos numéricos , Humanos , Internet , Neoplasias/inmunología , Neoplasias/prevención & control , Proteínas/inmunología , Programas Informáticos , Vacunas Virales/inmunología , Virosis/inmunología , Virosis/prevención & control , Virus/inmunologíaRESUMEN
In a previous study, we found that the global genome organizer Special AT-rich binding protein 1 (SATB1) is highly expressed in mesenchymal-derived human osteosarcoma U2OS cells and that the knock-down of SATB1 results in the inhibition of cell proliferation. The present study was aimed at investigating the effect of silencing SATB1 on cell migration, invasion, apoptosis and resistance to the chemotherapeutic drug arsenic trioxide. Cell migration and invasion were detected by wound-healing assays and trans-well invasion assays, respectively. Cell apoptosis was analyzed by an in situ Cell Death Detection POD Kit, based on terminal deoxynucleotydyl transferase mediated dUTP nick-end labeling (TUNEL) staining and mRNAs were analyzed by real time qRT-PCR. We found that cell migration and invasion were inhibited and that the proportion of apoptotic cells and sensitivities to the chemotherapeutic drug arsenic trioxide were enhanced by knockdown of SATB1 in U2OS cells. Furthermore, mRNA of ABCC1 and ABCG2 were decreased strikingly after SATB1 silencing. It was concluded that the elevated expression of SATB1 in U2OS cells contributes to maintenance of the malignant phenotype and resistance to chemotherapeutic drugs ATO, suggesting that silencing SATB1 in the cells might improve the effects of arsenic trioxides in the treatment of osteosarcoma in which SATB1 is over-expressed and that ABCC1 and ABCG2 were involved in SATB1 mediated resistance of U2OS cells to ATO.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Neoplasias Óseas/terapia , Proteínas de Unión a la Región de Fijación a la Matriz/antagonistas & inhibidores , Osteosarcoma/terapia , Óxidos/farmacología , Apoptosis/genética , Trióxido de Arsénico , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Resistencia a Antineoplásicos/genética , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Invasividad Neoplásica/genética , Osteosarcoma/genética , Osteosarcoma/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Peritrophic membranes (PMs) are composed of chitin and protein. Chitin and protein play important roles in the structural formation and function of the PM. A new type I PM protein, HoCBP76, was identified from the Holotrichia oblita. HoCBP76 was shown as a 62.3 kDa protein by SDS-PAGE analysis and appeard to be associated with the PM throughout its entire length. In H. oblita larvae, the midgut is the only tissue where HoCBP76 could be detected during the feeding period of the larvae. The predicted amino acid sequence indicates that it contains seven tandem chitin binding domains belonging to the peritrophin-A family. HoCBP76 has chitin binding activity and is strongly associated with the PM. The HoCBP76 was not a mucin-like glycoprotein, and the consensus of conserved cysteines appeared to be CX13-17CX5CX9CX12CX7C. Western blot analysis showed that the abundance of HoCBP76 in the anterior, middle and posterior regions of the midgut was similar, indicating that HoCBP76 was secreted by the whole midgut epithelium, and confirmed the H. oblita PM belonged to the Type I PM. Immunolocalization analysis showed that HoCBP76 was mainly localized in the PM. The HoCBP76 is the first PM protein found in the H. oblita; however, its biochemical and physiological functions require further investigation.
Asunto(s)
Quitina/metabolismo , Escarabajos/metabolismo , Proteínas de Insectos/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Quitina/química , Escarabajos/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Proteínas de Insectos/química , Proteínas de Insectos/genética , Larva/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de SecuenciaRESUMEN
The PTEN gene negatively regulates the oncogenic PI3K-AKT pathway by encoding a lipid and protein phosphatase that dephosphorylates lipid phosphatidylinositol-3,4,5-triphosphate (PIP3) resulting in the inhibition of PI3K and downstream inhibition of AKT. Overexpression of PTEN in mice leads to a longer lifespan compared to control littermates, although the mechanism is unknown. Here, we provide evidence that young adult PTENOE mice exhibit many characteristics shared by other slow-aging mouse models, including those with mutations that affect GH/IGF1 pathways, calorie-restricted mice, and mice treated with anti-aging drugs. PTENOE white adipose tissue (WAT) has increased UCP1, a protein linked to increased thermogenesis. WAT of PTENOE mice also shows a change in polarization of fat-associated macrophages, with elevated levels of arginase 1 (Arg1, characteristic of M2 macrophages) and decreased production of inducible nitric oxide synthase (iNOS, characteristic of M1 macrophages). Muscle and hippocampus showed increased expression of the myokine FNDC5, and higher levels of its cleavage product irisin in plasma, which has been linked to increased conversion of WAT to more thermogenic beige/brown adipose tissue. PTENOE mice also have an increase, in plasma and liver, of GPLD1, which is known to improve cognition in mice. Hippocampus of the PTENOE mice has elevation of both BDNF and DCX, indices of brain resilience and neurogenesis. These changes in fat, macrophages, liver, muscle, hippocampus, and plasma may be considered "aging rate indicators" in that they seem to be consistently changed across many of the long-lived mouse models and may help to extend lifespan by delaying many forms of late-life illness. Our new findings show that PTENOE mice can be added to the group of long-lived mice that share this multi-tissue suite of biochemical characteristics.
Asunto(s)
Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Animales , Ratones , Envejecimiento , Fibronectinas/metabolismo , Lípidos , Fenotipo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genéticaRESUMEN
Dietary restriction (DR) and hypoxia (low oxygen) extend lifespan in Caenorhabditis elegans through the induction of a convergent downstream longevity gene, fmo-2. Flavin-containing monooxygenases (FMOs) are highly conserved xenobiotic-metabolizing enzymes with a clear role in promoting longevity in nematodes and a plausible similar role in mammals. This makes them an attractive potential target of small molecule drugs to stimulate the health-promoting effects of longevity pathways. Here, we utilize an fmo-2 fluorescent transcriptional reporter in C. elegans to screen a set of 80 compounds previously shown to improve stress resistance in mouse fibroblasts. Our data show that 19 compounds significantly induce fmo-2, and 10 of the compounds induce fmo-2 more than twofold. Interestingly, 9 of the 10 high fmo-2 inducers also extend lifespan in C. elegans. Two of these drugs, mitochondrial respiration chain complex inhibitors, interact with the hypoxia pathway to induce fmo-2, whereas two dopamine receptor type 2 (DRD2) antagonists interact with the DR pathway to induce fmo-2, indicating that dopamine signaling is involved in DR-mediated fmo-2 induction. Together, our data identify nine drugs that each (1) increase stress resistance in mouse fibroblasts, (2) induce fmo-2 in C. elegans, and (3) extend nematode lifespan, some through known longevity pathways. These results define fmo-2 induction as a viable approach to identifying and understanding mechanisms of putative longevity compounds.
Asunto(s)
Caenorhabditis elegans , Longevidad , Animales , Caenorhabditis elegans/efectos de los fármacos , Longevidad/efectos de los fármacos , Ratones , Oxigenasas/metabolismo , Oxigenasas/genética , Restricción Calórica , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Evaluación Preclínica de Medicamentos/métodosRESUMEN
Detoxification of ingested xenobiotic chemicals, and of potentially toxic endogenous metabolites, is carried out largely through a series of enzymes synthesized in the liver, sometimes called "xenobiotic metabolizing enzymes" (XME). Expression of these XME is sexually dimorphic in rodents and humans, with many of the XME expressed at higher levels in females. This expression pattern is thought to be regulated, in part, by the sex differences in circadian growth hormone (GH) pulsatility. We have evaluated mRNA, in the liver, for 52 XME genes in male and female mice of four mutant stocks, with diminished levels of GH receptor (GHR) either globally (GKO), or in liver (LKO), fat (FKO), or muscle (MKO) tissue specifically. The data show complex, sex-specific changes. For some XME, the expression pattern is consistent with direct control of hepatic mRNA by GHR in the liver. In contrast, other XME show evidence for indirect pathways in which hepatic XME expression is altered by GH signals in fat or skeletal muscle. The effects of GHR-null mutations on glucose control, responses to dietary interventions, steroid metabolism, detoxification pathways, and lifespan may depend on a mixture of direct hepatic effects and cross talk between different GH-responsive tissues.