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In this issue of Molecular Cell, Horvathova et al. (2017) have developed a powerful approach to single-molecule assessment of RNA decay in living cells by exploiting the ability of flavivirus RNA structural elements to trap XRN1 decay intermediates in dual-labeled reporter constructs.
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Exorribonucleasas , Imagen Individual de Molécula , Estabilidad del ARN , ARN MensajeroRESUMEN
Multisource feedback has long been a recommended tool to assess clinical competencies within graduate medical education. Additionally, incorporating feedback supplied by patients and other members of the healthcare team can provide the framework to bridge perspectives and viewpoints that may be different from their own. This, in effect, can aid in fortifying values in diversity, equity, and inclusivity by developing more knowledgeable, empathetic, and respectful future healthcare providers.
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N6-methyladenosine (m6A) is an abundant post-transcriptional modification that can impact RNA fate via interactions with m6A-specific RNA binding proteins. Despite accumulating evidence that m6A plays an important role in modulating pluripotency, the influence of m6A reader proteins in pluripotency is less clear. Here, we report that YTHDF2, an m6A reader associated with mRNA degradation, is highly expressed in induced pluripotent stem cells (iPSCs) and down-regulated during neural differentiation. Through RNA sequencing, we identified a group of m6A-modified transcripts associated with neural development that are directly regulated by YTDHF2. Depletion of YTHDF2 in iPSCs leads to stabilization of these transcripts, loss of pluripotency, and induction of neural-specific gene expression. Collectively, our results suggest YTHDF2 functions to restrain expression of neural-specific mRNAs in iPSCs and facilitate their rapid and coordinated up-regulation during neural induction. These effects are both achieved by destabilization of the targeted transcripts.
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Adenosina/análogos & derivados , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Células-Madre Neurales/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Células-Madre Neurales/citología , ARN Mensajero/química , Proteínas de Unión al ARN/fisiologíaRESUMEN
GPR6 is an orphan G-protein-coupled receptor that has enriched expression in the striatopallidal, indirect pathway and medium spiny neurons of the striatum. This pathway is greatly impacted by the loss of the nigro-striatal dopaminergic neurons in Parkinson disease, and modulating this neurocircuitry can be therapeutically beneficial. In this study, we describe the in vitro and in vivo pharmacological characterization of (R)-1-(2-(4-(2,4-difluorophenoxy)piperidin-1-yl)-3-((tetrahydrofuran-3-yl)amino)-7,8-dihydropyrido[3,4-b]pyrazin-6(5H)-yl)ethan-1-one (CVN424), a highly potent and selective small-molecule inverse agonist for GPR6 that is currently undergoing clinical evaluation. CVN424 is brain-penetrant and shows dose-dependent receptor occupancy that attained brain 50% of receptor occupancy at plasma concentrations of 6.0 and 7.4 ng/ml in mice and rats, respectively. Oral administration of CVN424 dose-dependently increases locomotor activity and reverses haloperidol-induced catalepsy. Furthermore, CVN424 restored mobility in bilateral 6-hydroxydopamine lesion model of Parkinson disease. The presence and localization of GPR6 in medium spiny neurons of striatum postmortem samples from both nondemented control and patients with Parkinson disease were confirmed at the level of both RNA (using Nuclear Enriched Transcript Sort sequencing) and protein. This body of work demonstrates that CVN424 is a potent, orally active, and brain-penetrant GPR6 inverse agonist that is effective in preclinical models and is a potential therapeutic for improving motor function in patients with Parkinson disease. SIGNIFICANCE STATEMENT: CVN424 represents a nondopaminergic novel drug for potential use in patients with Parkinson disease.
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Cuerpo Estriado , Animales , Hormonas Esteroides Gonadales , RatasRESUMEN
Insect-borne flaviviruses produce a 300-500-base long noncoding RNA, termed subgenomic flavivirus RNA (sfRNA), by stalling the cellular 5'-3'-exoribonuclease 1 (XRN1) via structures located in their 3' UTRs. In this study, we demonstrate that sfRNA production by Zika virus represses XRN1 analogous to what we have previously shown for other flaviviruses. Using protein-RNA reconstitution and a stringent RNA pulldown assay with human choriocarcinoma (JAR) cells, we demonstrate that the sfRNAs from both dengue type 2 and Zika viruses interact with a common set of 21 RNA-binding proteins that contribute to the regulation of post-transcriptional processes in the cell, including splicing, RNA stability, and translation. We found that four of these sfRNA-interacting host proteins, DEAD-box helicase 6 (DDX6) and enhancer of mRNA decapping 3 (EDC3) (two RNA decay factors), phosphorylated adaptor for RNA export (a regulator of the biogenesis of the splicing machinery), and apolipoprotein B mRNA-editing enzyme catalytic subunit 3C (APOBEC3C, a nucleic acid-editing deaminase), inherently restrict Zika virus infection. Furthermore, we demonstrate that the regulations of cellular mRNA decay and RNA splicing are compromised by Zika virus infection as well as by sfRNA alone. Collectively, these results reveal the large extent to which Zika virus-derived sfRNAs interact with cellular RNA-binding proteins and highlight the potential for widespread dysregulation of post-transcriptional control that likely limits the effective response of these cells to viral infection.
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Estabilidad del ARN/fisiología , ARN no Traducido/metabolismo , Virus Zika/genética , Regiones no Traducidas 3' , Animales , Chlorocebus aethiops , ARN Helicasas DEAD-box/metabolismo , Exorribonucleasas/metabolismo , Flavivirus/genética , Genoma Viral/genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Conformación de Ácido Nucleico , Proteínas Proto-Oncogénicas/metabolismo , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , ARN no Traducido/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Células Vero , Virus Zika/metabolismo , Infección por el Virus Zika/virologíaRESUMEN
Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mechanisms associated with numerous post-transcriptional RNA processes. Historically, it has been difficult to examine all aspects of RNA decay in such an in vitro setting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present an approach to more confidently examine RNA decay parameters of large mRNA size transcripts through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing a 67 nucleotide xrRNA near the 3' end of any in vitro transcribed RNA with variable size or sequence context, investigators can observe the accumulation of the xrRNA as a readout of exoribonuclease-mediated 5'-3' decay. This approach may allow in vitro RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.
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Ingeniería Genética/métodos , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Estabilidad del ARN , ARN Mensajero/genética , ARN Viral/genética , Sistema Libre de Células , Electroforesis en Gel de Gradiente Desnaturalizante , Virus del Dengue/química , Virus del Dengue/genética , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Transcripción GenéticaRESUMEN
Changes in the rate of mRNA decay are closely coordinated with transcriptional changes and together these events have profound effects on gene expression during development and disease. Traditional approaches to assess mRNA decay have relied on inhibition of transcription, which can alter mRNA decay rates and confound interpretation. More recently, metabolic labeling combined with chemical modification and fractionation of labeled RNAs has allowed the isolation of nascent transcripts and the subsequent calculation of mRNA decay rates. This approach has been widely adopted for measuring mRNA half-lives on a global scale, but has proven challenging to use for analysis of single genes. We present a series of normalization and quality assurance steps to be used in combination with 4-thiouridine pulse labeling of cultured eukaryotic cells. Importantly, we demonstrate how the relative amount of 4sU-labeled nascent RNA influences accurate quantification. The approach described facilitates reproducible measurement of individual mRNA half-lives using 4-thiouridine and could be adapted for use with other nucleoside analogs.
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Marcadores de Afinidad/química , Estabilidad del ARN , ARN Mensajero/química , Coloración y Etiquetado/métodos , Tiouridina/química , Transcripción Genética , Animales , Biotinilación/métodos , Línea Celular , Células Eucariotas/metabolismo , Semivida , Humanos , Ratones , Control de Calidad , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/instrumentación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Coloración y Etiquetado/instrumentaciónRESUMEN
Sclerotinia stem rot (SSR) epidemics in soybean, caused by Sclerotinia sclerotiorum, are currently responsible for annual yield reductions in the United States of up to 1 million metric tons. In-season disease management is largely dependent on chemical control but its efficiency and cost-effectiveness depends on both the chemistry used and the risk of apothecia formation, germination, and further dispersal of ascospores during susceptible soybean growth stages. Hence, accurate prediction of the S. sclerotiorum apothecial risk during the soybean flowering period could enable farmers to improve in-season SSR management. From 2014 to 2016, apothecial presence or absence was monitored in three irrigated (n = 1,505 plot-level observations) and six nonirrigated (n = 2,361 plot-level observations) field trials located in Iowa (n = 156), Michigan (n = 1,400), and Wisconsin (n = 2,310), for a total of 3,866 plot-level observations. Hourly air temperature, relative humidity, dew point, wind speed, leaf wetness, and rainfall were also monitored continuously, throughout the season, at each location using high-resolution gridded weather data. Logistic regression models were developed for irrigated and nonirrigated conditions using apothecial presence as a binary response variable. Agronomic variables (row width) and weather-related variables (defined as 30-day moving averages, prior to apothecial presence) were tested for their predictive ability. In irrigated soybean fields, apothecial presence was best explained by row width (r = -0.41, P < 0.0001), 30-day moving averages of daily maximum air temperature (r = 0.27, P < 0.0001), and daily maximum relative humidity (r = 0.16, P < 0.05). In nonirrigated fields, apothecial presence was best explained by using moving averages of daily maximum air temperature (r = -0.30, P < 0.0001) and wind speed (r = -0.27, P < 0.0001). These models correctly predicted (overall accuracy of 67 to 70%) apothecial presence during the soybean flowering period for four independent datasets (n = 1,102 plot-level observations or 30 daily mean observations).
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Ascomicetos/fisiología , Producción de Cultivos/métodos , Glycine max , Enfermedades de las Plantas/microbiología , Tiempo (Meteorología) , Ascomicetos/crecimiento & desarrollo , Iowa , Modelos Logísticos , Michigan , Riesgo , Glycine max/crecimiento & desarrollo , Esporas Fúngicas/fisiología , WisconsinRESUMEN
The MAPK signaling cascade, comprised of several linear and intersecting pathways, propagates signaling into the nucleus resulting in cytokine and chemokine release. The Map Kinase Kinase isoforms 3 and 6 (MKK3 and MKK6) are responsible for the phosphorylation and activation of p38, and are hypothesized to play a key role in regulating this pathway without the redundancy seen in downstream effectors. Using FBDD, we have discovered efficient and selective inhibitors of MKK3 and MKK6 that can serve as tool molecules to help further understand the role of these kinases in MAPK signaling, and the potential impact of inhibiting kinases upstream of p38.
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Diseño de Fármacos , MAP Quinasa Quinasa 3/antagonistas & inhibidores , MAP Quinasa Quinasa 6/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Sitios de Unión , Humanos , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Células U937 , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The Puf family of RNA-binding proteins regulates gene expression primarily by interacting with the 3' untranslated region (3' UTR) of targeted mRNAs and inhibiting translation and/or stimulating decay. Physical association and computational analyses of yeast Puf3p identified >150 potential mRNA targets involved in mitochondrial function. However, only COX17 has been established as a target of Puf3p-mediated deadenylation and decapping. We have identified 10 new targets that are rapidly degraded in a Puf3p-dependent manner. We also observed changes in Puf3p activity in response to environmental conditions. Puf3p promotes rapid degradation of mRNA targets in the fermentable carbon source dextrose. However, Puf3p-mediated decay activity is inhibited in carbon sources that require mitochondrial function for efficient cell growth. In addition, the activity of Puf3p is rapidly altered by changing the carbon source. PUF3 expression is not decreased at the RNA or protein level by different carbon sources and localization is not significantly altered, suggesting that Puf3p activity is regulated posttranslationally. Finally, under conditions when Puf3p is unable to stimulate decay, Puf3p can still bind its target mRNAs. Together, these experiments provide insight into the carbon source-specific control of Puf3p activity and how such alterations allow Puf3p to dynamically regulate mitochondrial function.
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Carbono/metabolismo , Proteínas Mitocondriales/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Regiones no Traducidas 3' , Liasas/genética , Liasas/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Coronavirus disease 2019 (COVID-19) infection has been associated with a multitude of complications, one established complication being thromboembolism, a result of the proinflammatory state induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This prothrombotic state is a cumulation of many inflammatory pathways at work. Here, we present an interesting case of a 43-year-old female who did not present with the typical COVID-19 clinical picture. Instead, she presented with periumbilical pain, nausea, and vomiting. Upon further investigation, she was found to have a splenic infarct on a computed tomography (CT) scan. An extensive workup was performed to explore possible etiologies; however, it was concluded that her splenic infarct was secondary to her COVID-19 infection. With this case, we aim to add to the literature regarding the manifestations of the prothrombotic state of SARS-CoV-2.
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We present a fascinating case of a patient who suffered from persistent headaches for three months due to an epidermoid cyst located in the prepontine cistern. Epidermoid cysts are a very uncommon type of intracranial tumor, known for their slow growth and gradual onset of neurological symptoms. In this particular case, our patient, a 35-year-old, experienced a headache that was accompanied by dizziness, photophobia, and pain when moving their eyes. Further imaging revealed a cystic lesion in the prepontine cistern, which had a mass effect on the pons. After confirming the lesion was likely an epidermoid cyst through an MRI, the patient underwent surgery to have it removed. We hope to highlight the rarity of this type of tumor and its unique features when viewed through imaging.
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The lung's sarcomatoid carcinomas (SC) are a heterogeneous sporadic group of non-small cell lung carcinomas (NSCLCs) and are very challenging to diagnose and treat. Spindle cell carcinoma (SpCC) is a very rare subset of this group. Hence, the prognosis and treatments are unclear due to the limited literature available. The presentation of this cancer varies based on the site of the neoplasm and the complications and metastases observed at the time of diagnosis. Here, we report a 73-year-old man who presented to the emergency room after two months of worsening dyspnea and fatigue. Chest X-ray showed an extensive left-sided pleural effusion. A computed tomography (CT) scan of the chest showed a pleural-based mass that came back as SpCC, for which he was referred to a university hospital.
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The online community resource Phytophthora database (PD) was developed to support accurate and rapid identification of Phytophthora and to help characterize and catalog the diversity and evolutionary relationships within the genus. Since its release in 2008, the sequence database has grown to cover 1 to 12 loci for ≈2,600 isolates (representing 138 described and provisional species). Sequences of multiple mitochondrial loci were added to complement nuclear loci-based phylogenetic analyses and diagnostic tool development. Key characteristics of most newly described and provisional species have been summarized. Other additions to improve the PD functionality include: (i) geographic information system tools that enable users to visualize the geographic origins of chosen isolates on a global-scale map, (ii) a tool for comparing genetic similarity between isolates via microsatellite markers to support population genetic studies, (iii) a comprehensive review of molecular diagnostics tools and relevant references, (iv) sequence alignments used to develop polymerase chain reaction-based diagnostics tools to support their utilization and new diagnostic tool development, and (v) an online community forum for sharing and preserving experience and knowledge accumulated in the global Phytophthora community. Here we present how these improvements can support users and discuss the PD's future direction.
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Bases de Datos Genéticas , Phytophthora/genética , Biología Computacional , ADN Mitocondrial/genética , Bases de Datos Genéticas/tendencias , Genotipo , Geografía , Internet , Repeticiones de Microsatélite/genética , Filogenia , Phytophthora/clasificación , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Genetic approaches are limited in the dinoflagellate family, Symbiodiniaceae, causing a bottleneck in the discovery of useful mutants toward the goal of preventing future coral bleaching events. In this protocol, we demonstrate the application of UV exposure, coupled with downstream phenotypic screening and mutant isolation, to form a UV mutagenesis pipeline. This pipeline provides an avenue to generate Symbiodiniaceae mutants to help link genotype to phenotype, as well as address previously unanswered questions surrounding relationships with host organisms, like coral. For complete details on the use and execution of this protocol, please refer to Jinkerson et al. (2022).1.
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Dinoflagelados , Dinoflagelados/genética , Genotipo , Fenotipo , Mutagénesis/genéticaRESUMEN
Puf3p regulates the stability of nuclear-encoded mRNAs acting in mitochondrial biogenesis and function in Saccharomyces cerevisiae. This work identifies the phosphorylation of Pop2p, a component of the deadenylase complex, as being critical for adapting Puf3p-mediated mRNA decay upon carbon source alterations. We demonstrate that the Puf3p-Pop2p association diminishes in mitochondria-reliant conditions and establish Yak1p, a kinase that phosphorylates Pop2p at threonine 97, as a new player in Puf3p-mediated regulation of mRNA decay. Yak1p deletion alters the half-life of Puf3p target mRNAs. Our findings outline a metabolism-driven regulatory switch, whereby, in mitochondria-independent conditions, Puf3p recruits Pop2p and the decay machinery to bound mRNAs for rapid decay. Conversely, in mitochondria-reliant conditions, the association of Puf3p with Yak1p increases, placing Yak1p proximal to neighboring Pop2p. Subsequent Pop2p phosphorylation reduces the Puf3p-Pop2p interaction and stabilizes Puf3p target mRNAs.
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Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Carbono/metabolismo , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Photosynthesis shapes the symbiotic relationships between cnidarians and Symbiodiniaceae algae-with many cnidarian hosts requiring symbiont photosynthate for survival-but little is known about how photosynthesis impacts symbiosis establishment. Here, we show that during symbiosis establishment, infection, proliferation, and maintenance can proceed without photosynthesis, but the ability to do so is dependent on specific cnidarian-Symbiodiniaceae relationships. The evaluation of 31 pairs of symbiotic relationships (five species of Symbiodiniaceae in sea anemone, coral, and jellyfish hosts) revealed that infection can occur without photosynthesis. A UV mutagenesis method for Symbiodiniaceae was established and used to generate six photosynthetic mutants that can infect these hosts. Without photosynthesis, Symbiodiniaceae cannot proliferate in the sea anemone Aiptasia or jellyfish Cassiopea but can proliferate in the juvenile polyps of the coral Acropora. After 6 months of darkness, Breviolum minutum is maintained within Aiptasia, indicating that Symbiodiniaceae maintenance can be independent of photosynthesis. Manipulating photosynthesis provides insights into cnidarian-Symbiodiniaceae symbiosis.
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Antozoos , Dinoflagelados , Anémonas de Mar , Animales , Fotosíntesis , SimbiosisRESUMEN
The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 is a target for virus neutralizing antibodies. The V3 sequence determines whether the virus will manifest R5 or X4 phenotypes and use the CCR5 or CXCR4 chemokine coreceptor, respectively. Previous NMR studies revealed that both R5- and X4-V3 peptides bound to antibodies 0.5beta and 447-52D form beta-hairpin conformations with the GPGR segment at the turn. In contrast, in their free form, linear V3 peptides and a cyclic peptide consisting of the entire 35-residue V3 loop were highly unstructured in aqueous solution. Herein we evaluated a series of synthetic disulfide constrained V3-peptides in which the position of the disulfide bonds, and therefore the ring size, was systematically varied. NMR structures determined for singly and doubly disulfide constrained V3-peptides in aqueous solution were compared with those found for unconstrained V3(JRFL) and V3(IIIB) peptides bound to 447-52D and to 0.5beta, respectively. Our study indicated that cyclic V3 peptides manifested significantly reduced conformational space compared to their linear homologues and that in all cases cyclic peptides exhibited cross-strand interactions suggestive of beta-hairpin-like structures. Nevertheless, the singly constrained V3-peptides retained significant flexibility and did not form an idealized beta-hairpin. Incorporation of a second disulfide bond results in significant overall rigidity, and in one case, a structure close to that of V3(MN) peptide bound to 447-52D Fab was assumed and in another case a structure close to that formed by the linear V3(IIIB) peptide bound to antibody 0.5beta was assumed.
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Anticuerpos Antivirales/metabolismo , Sitios de Unión de Anticuerpos , Epítopos/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/metabolismo , Imitación Molecular , Secuencia de Aminoácidos , Anticuerpos Antivirales/química , Disulfuros , Epítopos/inmunología , Epítopos/metabolismo , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/inmunología , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Pruebas de Neutralización , Péptidos/síntesis química , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMEN
Phytophthora spp. represent a serious threat to agricultural and ecological systems. Many novel Phytophthora spp. have been reported in recent years, which is indicative of our limited understanding of the ecology and diversity of Phytophthora spp. in nature. Systematic cataloging of genotypic and phenotypic information on isolates of previously described species serves as a baseline for identification, classification, and risk assessment of new Phytophthora isolates. The Phytophthora Database (PD) was established to catalog such data in a web-accessible and searchable format. To support the identification of new Phytophthora isolates via comparison of their sequences at one or more loci with the corresponding sequences derived from the isolates archived in the PD, we generated and deposited sequence data from more than 1,500 isolates representing the known diversity in the genus. Data search and analysis tools in the PD include BLAST, Phyloviewer (a program for building phylogenetic trees using sequences of selected isolates), and Virtual Gel (a program for generating expected restriction patterns for given sequences). The PD also provides a customized means of storing and sharing data via the web. The PD serves as a model that easily can be adopted to develop databases for other important pathogen groups.