RESUMEN
Salt is a major environmental stress factor that can affect rice growth and yields. Recent studies suggested that members of the AP2/ERF domain-containing RAV (related to ABI3/VP1) TF family are involved in abiotic stress adaptation. However, the transcriptional response of rice RAV genes (OsRAVs) to salt has not yet been fully characterized. In this study, the expression patterns of all five OsRAVs were examined under salt stress. Only one gene, OsRAV2, was stably induced by high-salinity treatment. Further expression profile analyses indicated that OsRAV2 is transcriptionally regulated by salt, but not KCl, osmotic stress, cold or ABA (abscisic acid) treatment. To elucidate the regulatory mechanism of the stress response at the transcriptional level, we isolated and characterized the promoter region of OsRAV2 (P OsRAV2 ). Transgenic analysis indicated that P OsRAV2 is induced by salt stress but not osmotic stress or ABA treatment. Serial 5' deletions and site-specific mutations in P OsRAV2 revealed that a GT-1 element located at position -664 relative to the putative translation start site is essential for the salt induction of P OsRAV2 . The regulatory function of the GT-1 element in the salt induction of OsRAV2 was verified in situ in plants with targeted mutations generated using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system. Taken together, our results indicate that the GT-1 element directly controls the salt response of OsRAV2. This study provides a better understanding of the putative functions of OsRAVs and the molecular regulatory mechanisms of plant genes under salt stress.
Asunto(s)
Oryza/genética , Proteínas de Plantas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Cloruro de Sodio/farmacología , Adaptación Fisiológica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Oryza/efectos de los fármacos , Oryza/fisiología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Tolerancia a la Sal , Estrés FisiológicoRESUMEN
Hydrogen-peroxide (H2O2)-induced growth of small-sized gold nanoparticles (AuNPs) is often implemented for H2O2 sensing and plasmonic immunoassay. In contrast, there is little-to-no information in the literature regarding the application of H2O2-inhibited aggregation of citrate-capped AuNPs. This study discloses that benzene-1,4-diboronic acid (BDBA) was effective in driving the aggregation of citrate-capped AuNPs through an interaction between α-hydroxycarboxylate of citrate and boronic acids of BDBA. The H2O2-mediated oxidation of BDBA resulted in the conversion of boronic acid groups to phenol groups. The oxidized BDBA was incapable of triggering the aggregation of citrate-capped AuNPs. Thus, the presence of H2O2 prohibited BDBA-induced aggregation of citrate-capped AuNPs. The BDBA-induced aggregation of citrate-capped AuNPs can be paired with the glucose oxidase (GOx)-glucose system to design a colorimetric probe for glucose. Moreover, a H2O2·BDBA·AuNP probe was integrated with sandwich immunoassay, biotinylated antibody, and avidin-conjugated GOx for the selective naked-eye detection of rabbit immunoglobulin G (IgG) and human-prostate-specific antigen (PSA). The lowest detectable concentrations of rabbit IgG and human PSA by the naked eye were down to 0.1 and 4 ng/mL, respectively. More importantly, the proposed plasmonic immunoassay allowed the naked-eye quantification of 0-10 ng/mL PSA at an interval of 2 ng/mL in plasma samples.
Asunto(s)
Ácidos Borónicos/química , Oro/química , Inmunoensayo/métodos , Nanopartículas del Metal/química , Antígeno Prostático Específico/sangre , Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Ácido Cítrico/química , Colorimetría , Glucosa/análisis , Glucosa Oxidasa/química , Glucosa Oxidasa/metabolismo , Humanos , Peróxido de Hidrógeno/análisis , Inmunoglobulina G/inmunologíaRESUMEN
Epstein-Barr virus (EBV) expresses two immediate-early proteins, Rta and Zta, which are key transcription factors that can form a complex with MCAF1 at Zta-responsive elements (ZREs) to synergistically activate several viral lytic genes. Our previous research indicated that RanBPM interacts with Rta and enhances Rta sumoylation. Here we showed that RanBPM binds to Zta in vitro and in vivo, and acts as an intermediary protein in Rta-Zta complex formation. The Rta-RanBPM-Zta complex was observed to bind with ZREs in the transcriptional activation of key viral genes, such as BHLF1 and BHRF1, while the introduction of RanBPM short hairpin RNA (shRNA) subsequently reduced the synergistic activity of Zta and Rta. RanBPM was found to enhance Zta-dependent transcriptional activity via the inhibition of Zta sumoylation. Interestingly, Z-K12R, a sumoylation-defective mutant of Zta, demonstrated transcriptional activation capabilities that were stronger than those of Zta and apparently unaffected by RanBPM modulation. Finally, RanBPM silencing inhibited the expression of lytic proteins. Taken together, these results shed light on the mechanisms by which RanBPM regulates Zta-mediated transcriptional activation, and point to an important role for RanBPM in EBV lytic progression.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/genética , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/genética , Unión Proteica , Transactivadores/genéticaRESUMEN
This study describes the synthesis of fluorescent polydopamine dots (PDs) through hydroxyl radical-induced degradation of polydopamine nanoparticles. The decomposition of polydopamine nanoparticles to fluorescent PDs was confirmed using transmission electron microscopy and dark-field microscopy. The analysis of PDs by using laser desorption/ionization time-of-flight mass spectrometry revealed that the PDs consisted of dopamine, 5,6-dihydroxyindole, and trihydroxyindole units. Oligomerization and self-assembly of these units produced a broad adsorption band, resulting in an excitation-wavelength-dependent emission behavior. The maximal fluorescence of PDs appeared at 440 nm with a quantum yield of 1.2%. The coordination between the catechol groups of PDs and ferric ions (Fe(3+)) quenched the fluorescence of PDs; the limit of detection at a signal-to-noise ratio of 3 for Fe(3+) was determined to be 0.3 µM. The presence of pyrophosphate switched on the fluorescence of the PD-Fe(3+) complexes. Compared to the other reported methods for sensing Fe(3+), PDs provided simple, low-cost, and reusable detection of Fe(3+).
Asunto(s)
Radical Hidroxilo/química , Indoles/química , Nanopartículas/química , Polímeros/química , Transporte de Electrón , Compuestos Férricos/química , Nanopartículas/metabolismo , Nanopartículas/ultraestructura , Teoría Cuántica , Espectrometría de FluorescenciaRESUMEN
Epstein-Barr virus (EBV) encodes a transcription factor, Rta, which is required to activate the transcription of EBV lytic genes. This study demonstrates that treating P3HR1 cells with a proteasome inhibitor, MG132, causes the accumulation of SUMO-Rta and promotes the expression of EA-D. GST pulldown and coimmunoprecipitation studies reveal that RNF4, a RING-domain-containing ubiquitin E3 ligase, interacts with Rta. RNF4 also targets SUMO-2-conjugated Rta and promotes its ubiquitination in vitro. Additionally, SUMO interaction motifs in RNF4 are important to the ubiquitination of Rta because the RNF4 mutant with a mutation at the motifs eliminates ubiquitination. The mutation of four lysine residues on Rta that abrogated SUMO-3 conjugation to Rta also decreases the enhancement of the ubiquitination of Rta by RNF4. This finding demonstrates that RNF4 is a SUMO-targeted ubiquitin E3 ligase of Rta. Finally, knockdown of RNF4 enhances the expression of Rta and EA-D, subsequently promoting EBV lytic replication and virions production. Results of this study significantly contribute to efforts to elucidate a SUMO-targeted ubiquitin E3 ligase that regulates Rta ubiquitination to influence the lytic development of EBV.
Asunto(s)
Infecciones por Virus de Epstein-Barr/metabolismo , Herpesvirus Humano 4/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Ubiquitinación , Replicación Viral/fisiología , Secuencias de Aminoácidos , Línea Celular , Infecciones por Virus de Epstein-Barr/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Inmediatas-Precoces/genética , Mutación , Proteínas Nucleares/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Gene transformation is an important method for improvement of plants into elite varieties. However, the possibility of gene flow between genetically modified (GM) crops and similar species is a serious public issue that may potentially endanger ecological stability. Cleistogamy is expected to be an ideal genetic tool for preventing transgene propagation from GM crops. A rice mutant, cl7(t), was created by ethyl methanesulfonate mutagenesis. The mutant exhibited cleistogamy, and had closed spikelets, reduced plant height, and altered morphology of the leaves, panicle, and seeds. Anatomical investigations revealed that the cl7(t) mutant contained more vascular bundles and thicker stems than the wild type, which increased the mechanical strength of its internodes, and anti-lodging ability. Further studies demonstrated that the force required to open the lemma and palea was higher in the cl7(t) mutant, and there was weak swelling ability in the lodicules, which leads to cleistogamy. Allelic analyses and complementation tests indicated that cl7(t) was a novel allele of dep2, a mutant that was previously reported to have similar panicle morphology. Sequence analysis showed that cl7(t) had a single nucleotide substitution (C to A) in the third exon that leads to a Ser substitution with a stop codon, giving a truncated DEP2 protein. Quantitative RT-PCR and in situ hybridization tests demonstrated that there was lower CL7(t) expression level in the spikelets and weaker CL7(t) signals in the lodicules of the cl7(t) mutant compared with wild type, which implies that CL7(t) might participate in the development of lodicules. To improve the agronomic traits of cl7(t) to fit the needs of field production, the cl7(t) mutant was crossed with an intermediate-type rice variety named Guanghui102, which bears some important agronomic traits, including increased grain numbers and high rate of seed setting. Through multi-generational pedigree selection, cleistogamy lines with improved economic traits were obtained, which can be used for the selection of ecologically safe GM rice varieties.
Asunto(s)
Oryza/genética , Fenotipo , Proteínas de Plantas/genética , Alelos , Clonación Molecular , Prueba de Complementación Genética , Oryza/anatomía & histología , Oryza/metabolismo , Proteínas de Plantas/metabolismo , PolinizaciónRESUMEN
BACKGROUND & PROBLEMS: Peripheral intravenous catheter insertion is a significant source of stress for preschoolers during hospitalization. An average of about 85% of pediatric patients at our general pediatric unit are preschoolers. An average 71% of these exhibit severe pain-related behavior during intravenous insertions. The factors influencing this pain experience may include inappropriate administration of analgesics by nurses, non-pharmacologic pain management, and inappropriate clinical settings. PURPOSE: This project worked to develop a strategy to reduce the incidence of severe injection pain in preschool children from 71.0% to 36.0% and to achieve a capacity improvement target of 50%. RESOLUTIONS: We implemented the following: 1) arranged a relevant training program for pediatric nurses; 2) revised hospital standards for pediatric intravenous insertions; and 3) enhanced analgesic administration and non-pharmacologic pain management through creating child-friendly clinical settings and providing interactive toys. RESULTS: After implementing the above mentioned interventions, the incidence of severe pain-related behavior in pediatric patients decreased from 71.0% to 19.7%, a result that greatly exceeded expectations. CONCLUSIONS: This project demonstrated an effective approach to reducing severe intravenous-insertion pain in preschoolers and increasing pediatric care quality.
Asunto(s)
Inyecciones Intravenosas/efectos adversos , Dolor/prevención & control , Preescolar , Femenino , Humanos , MasculinoRESUMEN
Mitochondrial retrograde regulation (MRR) is the transduction of mitochondrial signals to mediate nuclear gene expression. It is not clear whether MRR is a common regulation mechanism in plant abiotic stress response. In this study, we analysed the early abiotic stress response of the rice OsAOX1 genes, and the induction of OsAOX1a and OsAOX1b (OsAOX1a/b) was selected as a working model for the stress-induced MRR studies. We found that the induction mediated by the superoxide ion (O2·(-) )-generating chemical methyl viologen was stronger than that of hydrogen peroxide (H2 O2 ). The addition of reactive oxygen species (ROS) scavengers demonstrated that the stress induction was reduced by eliminating O2·(-) . Furthermore, the stress induction did not rely on chloroplast- or cytosol-derived O2·(-) . Next, we generated transgenic plants overexpressing the superoxide dismutase (SOD) gene at different subcellular locations. The results suggest that only the mitochondrial SOD, OsMSD, attenuated the stress induction of OsAOX1a/b specifically. Therefore, our findings demonstrate that abiotic stress initiates the MRR on OsAOX1a/b and that mitochondrial O2·(-) is involved in the process.
Asunto(s)
Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Oryza/fisiología , Oxidorreductasas/metabolismo , Paraquat/farmacología , Proteínas de Plantas/metabolismo , Transducción de Señal , Estrés Fisiológico , Calcio/metabolismo , Núcleo Celular/metabolismo , Frío , Sequías , Depuradores de Radicales Libres/farmacología , Expresión Génica , Regulación de la Expresión Génica de las Plantas , Peróxido de Hidrógeno/farmacología , Mitocondrias/fisiología , Proteínas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/genética , Oryza/efectos de los fármacos , Oryza/enzimología , Oryza/genética , Estrés Oxidativo , Oxidorreductasas/efectos de los fármacos , Oxidorreductasas/genética , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Especies Reactivas de Oxígeno/farmacología , Salinidad , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/análisis , Superóxidos/farmacologíaRESUMEN
Zta, encoded by the BZLF1 gene of Epstein-Barr virus (EBV), is a transcription factor that is expressed during the immediate-early stage of the lytic cycle. The expression of Zta is crucial to viral lytic development. Earlier studies showed that Ku80 is a binding partner of Zta in ZKO-293 cells and is co-purified with Zta. This study verifies the interaction between Ku80 and Zta by using glutathione S-transferase-pull-down and co-immunoprecipitation assays, and also by indirect immunofluorescence analysis. This investigation also reveals that Ku80 binds to Zta on Zta-response elements in the BHLF1 promoter, enhancing the promoter activity. This study also reveals that the interaction between Zta and Ku80 involves the C-terminal region of Zta and the 425 aa N-terminal region of Ku80. The interaction between these two proteins and the enhancement of transcription that is activated by Zta suggest that Ku80 is important to EBV lytic development.
Asunto(s)
Antígenos Nucleares/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Viral de la Expresión Génica , Herpesvirus Humano 4/fisiología , Interacciones Huésped-Patógeno , Transactivadores/metabolismo , Transcripción Genética , Línea Celular , ADN Viral/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoprecipitación , Autoantígeno Ku , Regiones Promotoras Genéticas , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice.
Asunto(s)
Genes de Plantas/genética , Oryza/genética , Almidón/química , Estudios de Asociación Genética , Endogamia , Fenotipo , Dinámica Poblacional , Reproducibilidad de los Resultados , Almidón/biosíntesis , ViscosidadRESUMEN
Epstein-Barr virus (EBV) encodes more than 40 miRNAs that target cellular mRNAs to aid its infection, replication, and maintenance in individual cells and in its human host. Importin-7 (IPO7), also termed Imp7 or RanBPM7, is a nucleocytoplasmic transport protein that has been frequently identified as a target for two of these viral miRNAs. How the viral life cycle might benefit from regulating IPO7 has been unclear, though. We demonstrate with CRISPR-Cas9 mutagenesis that IPO7 is essential in at least three cells lines and that increasing its levels of expression inhibits growth of infected cells. EBV thus regulates the level of IPO7 to limit its accumulation consistent with its being required for survival of its host cell.
RESUMEN
Cryopreservation is widely used in umbilical cord blood (UCB) banking, yet its impact on progenitor cell function remains largely unaddressed. It is unknown whether long-term cryopreservation affects UCB transplantation outcomes. Herein, we evaluated the impact of UCB age on clinical outcomes and investigated the effect of cryopreservation duration of UCB on hematopoietic potency in 91 patients receiving single cord blood transplantations. UCB cryopreservation duration was 0.7 to 13.4 y. The most common indication of transplant was thalassemia (48%). There was no significant association between cryopreservation duration and neutrophil engraftment probability ( P = 0.475). Cryopreservation duration did not affect the post-thaw viability and subsequent neutrophil engraftment rate. Therefore, UCB units can undergo cryopreservation for at least 8 y with no impact on clinical outcomes.
Asunto(s)
Bancos de Sangre , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Criopreservación/métodos , Supervivencia Celular/fisiología , Sangre Fetal/citología , HumanosRESUMEN
BACKGROUND/AIMS: To investigate the viral kinetics of Chinese CHC patients received pegylated interferon plus ribavirin and examine the impact of HCV genotypes and severity of liver disease. METHODOLOGY: 65 treatment-naove CHC patients who finished a 24-week therapy with peginterferon (alpha-2b (1.5 mcg/kg/week) plus ribavirin (1000-1200 mg /day) and 24 weeks of follow-up were enrolled. Hepatic fibrosis was graded by the METAVIR scoring system. Serum quantitative HCV RNA was determined by Versant HCV RNA 3.0 assay (Bayer Inc.). RESULTS: Genotype non-1 patients responded quickly and a higher percentage of them achieved undetectable HCV RNA (< 615 IU/mL) at week 4 compared with genotype 1 patients (93% vs. 69%, p = 0.018). Degree of hepatic fibrosis significantly affected end-of-treatment and sustained response (SVR). For patients who did not achieve early virological response (EVR), the negative predictive value for SVR was 100%. In genotype 1 patients, undetectable HCV RNA by week 4 was a good marker to predict treatment response, with a positive predictive value of 84% and a negative predictive value of 82%. CONCLUSIONS: EVR can be applied to Chinese patients as an early stopping rule. A 24-week duration of pegylated IFN/ribavirin might be adequate for genotype 1 patients who rapidly responded to therapy.
Asunto(s)
Hepacivirus/aislamiento & purificación , Hepatitis C/tratamiento farmacológico , Interferón-alfa/uso terapéutico , ARN Viral/sangre , Ribavirina/uso terapéutico , Pueblo Asiatico , Quimioterapia Combinada , Femenino , Genotipo , Hepatitis C/genética , Humanos , Interferón alfa-2 , Cinética , Masculino , Persona de Mediana Edad , Polietilenglicoles , Proteínas RecombinantesRESUMEN
Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe3O4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe3O4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H2O2) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe3O4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe2+/Fe3+ sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H2O2. Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe3O4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe3O4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe3O4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe3O4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe3O4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.
Asunto(s)
Nanopartículas del Metal , Adenosina Monofosfato , Compuestos Férricos , Peróxido de HidrógenoRESUMEN
Epstein-Barr virus (EBV) encodes multiple miRNAs known to contribute to its pathogenicity. Previous studies have found that the levels of some EBV miRNAs are 10-100 fold higher in biopsies and in tumor xenografts than in cells grown in culture. We have asked if these increased levels reflect transcriptional enhancement resulting from the tumor microenvironment, selection for increased levels of the EBV genome, or both. We measured the levels of BART miRNAs and their DNA templates in tumor xenografts induced from EBV-positive gastric carcinoma cells and EBV-negative gastric carcinoma cells expressing plasmid replicons encoding these miRNAs. We focused on BART miRNAs which are expressed in all tumors and found that they provide tumors selective growth advantages as xenografts. Stem-loop PCR and real-time PCR revealed that the xenografts expressed both higher levels of some miRNAs and viral DNA templates than did the corresponding cells in culture.
Asunto(s)
Carcinoma/genética , Infecciones por Virus de Epstein-Barr/genética , Herpesvirus Humano 4/genética , MicroARNs/genética , Neoplasias Gástricas/genética , Animales , Línea Celular Tumoral , ADN Viral/genética , Infecciones por Virus de Epstein-Barr/virología , Regulación Viral de la Expresión Génica , Genoma Viral/genética , Herpesvirus Humano 4/metabolismo , Humanos , Ratones , Trasplante de Neoplasias , Transcripción Genética/genética , Trasplante Heterólogo , Microambiente TumoralRESUMEN
Hospice and palliative care has been recognized as an essential part of emergency medicine; however, there is no consensus on the optimal model for the delivery of hospice and palliative care in the emergency department (ED). Therefore, we conducted a novel implementation in a tertiary medical center in Taiwan. In the preintervention period, we recruited a specialist for hospice and palliative medicine in the ED to lead our intervention. In the early stage of the intervention, starting on July 1, 2014, we encouraged and funded ED physicians and nurses to receive training for hospice and palliative medicine and residents of emergency medicine to rotate to the hospice ward. In the late stage of the intervention, we initiated educational programs in the ED, an interdisciplinary meeting with the hospice team every month, sharing information and experience via a cell phone communication app, and setting aside an emergency hospice room for end-of-life patients. We compared the outcomes among pre-, during, and postintervention periods. Compared with 4 in the preintervention period, the cases of do not resuscitate (DNR) per month increased significantly to 30.1 in the early stage of intervention, 23.9 in late stage of intervention, and 34.6 in the postintervention period (all Pâ<â.001 compared with the preintervention period). Compared with 10.8% in the preintervention period, the ratio of DNR orders signed in the ED/total DNR orders signed in the study hospital was increased to 17.1% in early stage of intervention, 12.5% in late stage of intervention, and 22.8% in postintervention. Compared with zero in preintervention and early intervention, the cases of consultation with the hospice team increased significantly to 19 cases per month in the late stage of intervention and postintervention. The ability of nurses in hospice and palliative care, including knowledge and the timing and method of consultation with the hospice team, was also significantly improved. We successfully implemented a novel model of hospice and palliative care in the ED via a champion, education, and close collaboration with the hospice team, which could be an important reference for other EDs and intensive care unit in the future.
Asunto(s)
Educación Médica , Servicio de Urgencia en Hospital , Cuidados Paliativos al Final de la Vida/métodos , Cuidados Paliativos/métodos , Anciano , Anciano de 80 o más Años , Conocimientos, Actitudes y Práctica en Salud , Humanos , Internado y Residencia/métodos , Persona de Mediana Edad , Aplicaciones Móviles , Modelos Teóricos , Enfermeras y Enfermeros , Grupo de Atención al Paciente , Médicos , Proyectos Piloto , Estudios Prospectivos , Derivación y Consulta/estadística & datos numéricos , Órdenes de Resucitación , Taiwán , Centros de Atención TerciariaRESUMEN
Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes.
Asunto(s)
Adenosina Trifosfato/química , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/química , Compuestos de Boro/química , Ensayo de Inmunoadsorción Enzimática/instrumentación , Hierro/química , Adenosina Trifosfato/efectos de la radiación , Compuestos de Boro/efectos de la radiación , Transporte de Electrón/efectos de la radiación , Diseño de Equipo , Análisis de Falla de Equipo , Hierro/efectos de la radiación , Luz , Nanoconjugados/química , Nanoconjugados/efectos de la radiaciónRESUMEN
Nitrogen recycling and redistribution are important for the environmental stress response of plants. In non-nitrogen-fixing plants, ureide metabolism is crucial to nitrogen recycling from organic sources. Various studies have suggested that the rate-limiting components of ureide metabolism respond to environmental stresses. However, the underlying regulation mechanism is not well understood. In this report, rice ureidoglycolate amidohydrolase (OsUAH), which is a recently identified enzyme catalyzing the final step of ureide degradation, was identified as low-temperature- (LT) but not abscisic acid- (ABA) regulated. To elucidate the LT regulatory mechanism at the transcriptional level, we isolated and characterized the promoter region of OsUAH (P OsUAH ). Series deletions revealed that a minimal region between -522 and -420 relative to the transcriptional start site was sufficient for the cold induction of P OsUAH . Detailed analyses of this 103-bp fragment indicated that a C-repeat/dehydration-responsive (CRT/DRE) element localized at position -434 was essential for LT-responsive expression. A rice C-repeat-binding factors/DRE-binding proteins 1 (CBFs/DREB1s) subfamily member, OsCBF3, was screened to specifically bind to the CRT/DRE element in the minimal region both in yeast one-hybrid assays and in in vitro gel-shift analysis. Moreover, the promoter could be exclusively trans-activated by the interaction between the CRT/DRE element and OsCBF3 in vivo. These findings may help to elucidate the regulation mechanism of stress-responsive ureide metabolism genes and provide an example of the member-specific manipulation of the CBF/DREB1 subfamily.
RESUMEN
The CRISPR/Cas9 system is becoming an important genome editing tool for crop breeding. Although it has been demonstrated that target mutations can be transmitted to the next generation, their inheritance pattern has not yet been fully elucidated. Here, we describe the CRISPR/Cas9-mediated genome editing of four different rice genes with the help of online target-design tools. High-frequency mutagenesis and a large percentage of putative biallelic mutations were observed in T0 generations. Nonetheless, our results also indicate that the progeny genotypes of biallelic T0 lines are frequently difficult to predict and that the transmission of mutations largely does not conform to classical genetic laws, which suggests that the mutations in T0 transgenic rice are mainly somatic mutations. Next, we followed the inheritance pattern of T1 plants. Regardless of the presence of the CRISPR/Cas9 transgene, the mutations in T1 lines were stably transmitted to later generations, indicating a standard germline transmission pattern. Off-target effects were also evaluated, and our results indicate that with careful target selection, off-target mutations are rare in CRISPR/Cas9-mediated rice gene editing. Taken together, our results indicate the promising production of inheritable and "transgene clean" targeted genome-modified rice in the T1 generation using the CRISPR/Cas9 system.
Asunto(s)
Sistemas CRISPR-Cas , Marcación de Gen , Genoma de Planta , Oryza/genética , Transgenes , Genes de Plantas , Inestabilidad Genómica , Patrón de Herencia , Mutagénesis , Mutación , Plantas Modificadas Genéticamente , Edición de ARNRESUMEN
Respiratory metabolism is an important though poorly understood facet of plant adaptation to stress. Posttranslational modification of aconitase, a component of the tricarboxylic acid cycle (TCA), may be involved in stress tolerance. However, such stress-related transcriptional regulation and its mechanism remain unknown. In this study, we found that expression of the rice Aconitase gene OsACO1 is induced in a time-dependent manner by heat but not other typical abiotic stresses. To analyze the transcriptional regulation mechanism underlying the response to heat, the OsACO1 promoter (POsACO1) was isolated and characterized in transgenic rice. Using qualitative and quantitative analyses, we found that the expression of the GUS reporter gene responded to heat in different tissues and at different stages of development when driven by POsACO1. A series of 5' distal deletions of POsACO1 was generated to delineate the region responsible for heat-induced gene expression. Transient expression analyses in tobacco leaves identified a 322-bp minimal region between -1386 and -1065 as being essential and sufficient for heat-induced expression by POsACO1. We screened for known heat response-related cis-elements in this 322-bp region; however, sequences correlating with heat-induced gene expression were not identified in POsACO1. Therefore, truncations and successive mutagenesis analyses were performed in this 322-bp region. By comparing the activities of promoter fragments and their derivatives, our results indicated that the heat response element resided in a 9-bp region between -1132 and -1124, a sequence that contains a W-box motif. Additional site-directed mutagenesis analyses eliminated the heat response activity of POsACO1 via the W-box element, and an electrophoretic mobility shift assay (EMSA) indicated the binding of POsACO1 by factors in the nuclear extracts of heat-stressed rice seedlings in a W-box-dependent manner. Our results illustrate the expression pattern of a key component of the TCA response to abiotic stress and establish a putative regulatory pathway in the transcriptional modulation of rice respiratory metabolism genes in response to heat.