RESUMEN
HIV-1 selectively packages two copies of its 5'-capped RNA genome (gRNA) during virus assembly, a process mediated by the nucleocapsid (NC) domain of the viral Gag polyprotein and encapsidation signals located within the dimeric 5' leader of the viral RNA. Although residues within the leader that promote packaging have been identified, the determinants of authentic packaging fidelity and efficiency remain unknown. Here, we show that a previously characterized 159-nt region of the leader that possesses all elements required for RNA dimerization, high-affinity NC binding, and packaging in a noncompetitive RNA packaging assay (ΨCES) is unexpectedly poorly packaged when assayed in competition with the intact 5' leader. ΨCES lacks a 5'-tandem hairpin element that sequesters the 5' cap, suggesting that cap sequestration may be important for packaging. Consistent with this hypothesis, mutations within the intact leader that expose the cap without disrupting RNA structure or NC binding abrogated RNA packaging, and genetic addition of a 5' ribozyme to ΨCES to enable cotranscriptional shedding of the 5' cap promoted ΨCES-mediated RNA packaging to wild-type levels. Additional mutations that either block dimerization or eliminate subsets of NC binding sites substantially attenuated competitive packaging. Our studies indicate that packaging is achieved by a bipartite mechanism that requires both sequestration of the 5' cap and exposure of NC binding sites that reside fully within the ΨCES region of the dimeric leader. We speculate that cap sequestration prevents irreversible capture by the cellular RNA processing and translation machinery, a mechanism likely employed by other viruses that package 5'-capped RNA genomes.
Asunto(s)
Regiones no Traducidas 5'/genética , Genoma Viral , VIH-1/genética , Caperuzas de ARN/metabolismo , ARN Viral/metabolismo , Virión/fisiología , Ensamble de Virus , Células HEK293 , Infecciones por VIH/virología , Humanos , Conformación de Ácido Nucleico , Caperuzas de ARN/química , Caperuzas de ARN/genética , ARN Viral/química , ARN Viral/genéticaRESUMEN
Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 µM, and that all high-affinity sites (Kd â² 400 nM) reside within a â¼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles' heel to which HIV therapeutics may be targeted.
Asunto(s)
Infecciones por VIH/virología , VIH-1/fisiología , Nucleocápside/metabolismo , ARN Viral , Secuencias Reguladoras de Ácido Ribonucleico , Ensamble de Virus , Secuencia de Bases , Sitios de Unión , Genoma Viral , Conformación de Ácido Nucleico , Proteínas de la Nucleocápside/metabolismo , Unión ProteicaRESUMEN
Acetamiprid insecticide has been widely used to control paddy insects. In order to find out the dissipation of acetamiprid residues in paddy (variety: Satabdi), field studies were conducted in Nadia, West Bengal. Acetamiprid (20% SP) was applied twice at 10 g (T1), 20 g (T2) and 40 g (T3) a.i. ha-1 with three replications along with untreated control (T4). Residue analysis of acetamiprid in paddy (leaf, grain, husk and straw) and soil was conducted utilizing high-pressure liquid chromatograph (HPLC) with UV detector at LOQ of 0.05 mg kg-1. The recoveries of acetamiprid from fortified paddy sample were obtained in the range of 81.8 to 93.1% (for leaf, grain, husk and straw), and for soil, it was 87.2 to 94.3% at the LOQ level and upper two levels of LOQ. The initial residue of acetamiprid (0.11-0.99 mg kg-1) dissipated following the first-order reaction kinetics with the half-life of 1.5 to 1.8 days in paddy leaf and 1.3 to 1.4 days in soil. In harvested samples of paddy straw, grain and soil, the residue was found below LOQ. Because of the rapid dissipation, acetamiprid may be considered to have low risk to the ecosystem. Therefore, the use of acetamiprid for plant protection in paddy may be considered safe for food and environmental health.
Asunto(s)
Monitoreo del Ambiente , Insecticidas/análisis , Residuos de Plaguicidas/análisis , Piridinas/análisis , Contaminantes del Suelo/análisis , Cromatografía Líquida de Alta Presión , Ecosistema , Semivida , Cinética , Neonicotinoides , Oryza/química , Suelo/químicaRESUMEN
Effective surveillance during smart cities' sustainable development allows their cleanliness to be maintained by reusing waste to produce renewable energy. This study quantifies the biodegradable waste generated in specific regions of several cities and presents ways to convert it into renewable energy. This energy can then be used to partially support regional energy demands. This study explores ways of reducing carbon emissions for biodegradable waste collection processes in regional centers, ultimately sending the biodegradable waste to the energy conversion center. The smart production system allows for the flexible production and autonomation of rates of conversion; green technology depends on each regional center's research management, which is a decision variable for reducing carbon emissions. The major contribution of this study is to consider an energy supply chain management with flexibility of energy conversion under the reduction of carbon emissions, which leads to a sustainable ESCM with the global maximum profit. This study uses mathematical modeling to decrease biodegradable waste with conversion of energy through a classical optimization technique. The solution to this mathematical model yielded significant results, providing insight into waste reduction, reduced carbon emissions and the conversion of biodegradable waste to energy. The model is examined using numerical experiments, and its conclusion supports the model with the fundamental assumptions. Results of sensitivity analysis provide insight into the reduction and re-utilization of wastes, carbon emission reduction, and the benefits of using renewable energy.
RESUMEN
The study demonstrates the comparative retention characteristics of chromium from aqueous environment on iron(III) cellulose nanocomposite bead under conventional (C-ads) and ultrasound assisted (U-ads) batch adsorption operation. Optimization of process parameters was achieved varying pH, contact/sonication time, initial Cr(VI) concentration, adsorbent dose at fixed solution temperature. Equilibrium was achieved within 5 h and 30 min of contact time in C-ads and U-ads respectively. Langmuir isotherm is found suitable in C-ads while Temkin in U-ads. Both the operation is aided by change in negative free energy, positive enthalpy and entropy. Ultrasound assisted adsorption is much favorable in terms of efficiency, feasibility, spontaneity and randomness. Both the operations follow more the second order than the first order kinetic model. The pore diffusion prevails more in case of U-ads while the surface adsorption in C-ads.
RESUMEN
A sensitive gas chromatographic method using a modified QuEChERS technique is reported for simultaneous determination, dissipation and risk assessment of fipronil and its metabolites in sugarcane and soil. Recoveries were 80.7-98.5% with precision within 1.4-16.5% estimated at the limits of detection (LOD) 0.0015-0.002⯵gâ¯g-1 and limits of quantification (LOQ) 0.005⯵gâ¯g-1. Fipronil dissipated with half-life (T1/2) of 2.8-4.3â¯days while for total fipronil it was 3.7-6.0â¯days following application of fipronil (5% SC) in sugarcane fields at recommended (100â¯g a.i. ha-1) and double the recommended (200â¯g a.i. ha-1) doses. Estimated pre-harvest intervals (PHI) for fipronil were 20.3-27.0â¯days in sugarcane plants, and for total fipronil the corresponding values were 28.2-37.8â¯days. No dietary risk was observed due to fipronil (RQdâ¯<â¯1) 5â¯days after application. Potential risk exists towards algae and soil macro-organism (RQsâ¯>â¯1), but for earthworms it was safe (RQsâ¯<â¯1).
Asunto(s)
Insecticidas/análisis , Residuos de Plaguicidas/análisis , Pirazoles/análisis , Saccharum/química , Cromatografía de Gases , Cromatografía de Gases y Espectrometría de Masas , Semivida , Humanos , Insecticidas/metabolismo , Límite de Detección , Hojas de la Planta/química , Hojas de la Planta/metabolismo , Pirazoles/metabolismo , Reproducibilidad de los Resultados , Medición de Riesgo , Saccharum/metabolismo , Contaminantes del Suelo/análisisRESUMEN
The study explored to develop an eco-friendly herbal fungicide from chloroform extract of Clerodendrum infortunatum leaves. The extract upon activity guided purification using flash chromatography yielded eight fractions F3 through F10. Total phenol and flavonoid contents in the fractions ranged from 0.12-48.25 mg GAE/g and 0.03-25.29 mg QE/g. LC-MS/MS analysis confirmed the identification of seven phenolic acids across different fractions, the total of which varied between 0 and 2.17 mg/g. Emulsifiable Concentrate (20%) formulation was made with the extract and fractions and tested against Phomopsis vexans causing fruit rot disease in brinjal. Of the various fractions, F8 displayed highest antifungal activity (ED50 = 46.8 µg/ml). Antifungal activity of leaf extract/fractions was correlated with total phenol, total flavonoid and total phenolic acids (r = -0.60 to -0.69). Among the phenolic acids, benzoic acid showed maximum antifungal activity followed by t-cinnamic acid. The relationship between phenolic composition and activity is also reported.
RESUMEN
The effects of different operational variables on the mechanistic function of laterite in removal of fluoride have been investigated. Thermodynamic parameters such as free energy change, enthalpy, and entropy of the process, as well as the sorption isotherm, were evaluated. The extent of solute removal is determined by initial solute concentration, operational conditions, laterite dose, and solution pH. For a fixed set of experimental conditions, a model equation is developed from which the percent removal corresponding to each load of fluoride is determined. The mechanism of fluoride adsorption is governed by the zero point charge of laterite and follows a first-order rate equation. pH has a vital role influencing the surface characteristics of laterite. To simulate the flow dynamics, fluoride solution was run through a fixed bed column. The pattern of breakthrough curves for different influent fluoride concentration, pH, and column bed height was characterized. The column efficiency was tested from the bed depth-service time model. The elution of the retained fluoride was studied and the effectiveness of column operation was determined by the retention-elution cycles.
Asunto(s)
Óxido de Aluminio/química , Fluoruros/química , Dióxido de Silicio/química , Contaminantes Químicos del Agua/química , Purificación del Agua/métodos , Abastecimiento de Agua/análisis , Adsorción , Compuestos de Calcio/química , Compuestos Férricos/química , Concentración de Iones de Hidrógeno , Cinética , Óxidos/química , Compuestos de Potasio/química , Compuestos de Sodio/química , Suelo , Propiedades de SuperficieRESUMEN
This work attempts to elucidate the effects of different operational variables affecting the mechanistic function of fly ash for removal of some priority organic pollutants viz. phenol and its analogues. Thermodynamic parameters like free energy change, enthalpy and entropy of the process, as well as the sorption isotherms for phenols on fly ash, were measured and the most suitable isotherm was determined. Results of the study indicate that the extent of solute removal is determined by the initial solute concentration, molecular size and molecular arrangement of the solute. At the fixed set of experimental conditions, a model equation can be developed from which the percent removal corresponding to the load of the particular solute is determined. It is assumed that the mechanism of adsorption is governed by the surface characteristics of fly ash; pH has a vital role in influencing the solute removal as both the ionizing power (acidity, pKa) of the solutes and the zero point charge of fly ash (pH(ZPC)) depend on the solution pH. Isotherm pattern and the free energy change indicate that the process is favorable, as well as spontaneous. The information gathered from the study will serve as a predictive modeling procedure for the analysis and design of the removal of organic pollutants and decontamination of water. The leaching experiment indicates that the retained solutes do not release from fly ash. The retained solutes can be recovered and utilized as industrial raw material.
Asunto(s)
Carbono/química , Modelos Teóricos , Fenol/química , Contaminantes Químicos del Agua/aislamiento & purificación , Agua/química , Adsorción , Ceniza del Carbón , Concentración de Iones de Hidrógeno , Residuos Industriales , Material Particulado , Factores de TiempoRESUMEN
Salicylaldoxime-immobilized silica gel was characterized and used as a potential sorbent for heavy metal ions, viz. Cu(II), Ni(II), Co(II), and Zn(II). The experimental conditions were optimized both in batch and column processes to achieve the maximum efficiency. Kinetic and thermodynamic parameters as well as isotherm constants were evaluated to test the feasibility of the process. The role of various metal ions and different anions were tested in order to monitor the process in case of real samples. The alkali metal, alkaline earth metal, and ammonium salts do not have any effect on the said process. This differential behavior can be effectively used for the decontamination of alkali metal, alkaline earth metal, and ammonium salts from Cu(II), Ni(II), Co(II), and Zn(II) ions via solid phase extraction following AAS measurement. The purification of the salts was confirmed by voltammetric experiment.
RESUMEN
The kinetic aspects of adsorption of some priority organic pollutants, viz., phenol (hydroxybenzene), o-hydroxyphenol (1,2-dihydroxybenzene), m-hydroxyphenol (1,3-dihydroxybenzene), and 4-nitrophenol (1-hydroxy-4-nitrobenzene), on fly ash have been studied. The process is found to be of complex nature consisting of both surface adsorption and pore diffusion, the extent being estimated from the diffusion coefficient value. Activation parameter data for the ultimate adsorption as well as the pore diffusion are also evaluated. The data indicate that in the studied solute concentration range, external transport mainly governs the rate-limiting process.
Asunto(s)
Modelos Químicos , Compuestos Orgánicos/química , Contaminantes Químicos del Agua , Difusión , Cinética , TermodinámicaRESUMEN
The cooperative influence of operational variables for fluoride adsorption on cerium loaded cellulose nanocomposite bead (CCNB) was assessed using Taguchi design tool. The percentage contribution of each operational variable was determined. The solution pH, with a maximum contribution of 80.78%, indicates its highest influence on the response, the adsorption percent of fluoride. The quality and validity of the experimental design were assessed from ANOVA and subsequently by the confirmation experiment. The equilibrium adsorption data showed that the Temkin isotherm is the most suited one compared to the Langmuir and Freundlich model. It is found that almost 90% adsorbed fluoride can be eluted with 0.01 (N) NaOH and the regenerated bead can successively be reused for at least three times.
RESUMEN
Despite the importance of accurately determining inorganic arsenic speciation in natural waters to predicting bioavailability and environmental and health impacts, there remains considerable debate about the most appropriate species preservation strategies to adopt. In particular, the high-iron, low-Eh (redox potential) shallow groundwaters in West Bengal, Bangladesh and SE Asia, the use of which for drinking and irrigation purposes has led to massive international concerns for human health, are particularly prone to changes in arsenic speciation after sampling. The effectiveness of HCl and EDTA preservation strategies has been compared and used on variably arsenic-rich West Bengali groundwater samples, analysed by ion chromatography-inductively coupled plasma-mass spectrometry (IC-ICP-MS). Immediate filtration and acidification with HCl followed by refrigerated storage was found to be the most effective strategy for minimizing the oxidation of inorganic As(III) during storage. The use of a PRP-X100 (Hamilton) column with a 20 mmol L(-1) NH4H2PO4 as mobile phase enabled the separation of Cl- from As(III), monomethylarsonic acid, dimethylarsinic acid and As(V), thereby eliminating any isobaric interference between 40Ar35Cl+ and 75As+. The use of EDTA as a preservative, whose action is impaired by the high calcium concentrations typical of these types of groundwater, resulted in marked oxidation during storage. The use of HCl is therefore indicated for analytical methods in which chloride-rich matrices are not problematical. The groundwaters analysed by IC-ICP-MS were found to contain between 5 and 770 ng As mL(-1) exclusively as inorganic arsenic species. As(III)/total-As varied between 0 and 0.94.
RESUMEN
The protozoan parasite Trypanosoma brucei undergoes a complex developmental cycle coordinated with cell cycle control. These processes in eukaryotes are frequently regulated through mitogen-activated protein kinases (MAPKs) and cyclin-dependent protein kinases (CDKs), respectively. We have discovered a novel protein kinase which shares features of both ERK-type MAPKs and CDKs (T. brucei ERK-like, CDK-like protein kinase). This molecule, named TbECK1, is similar to the unusual mammalian KKIAMRE protein kinase family. Moreover, TbECK1 possesses a long C-terminal extension reminiscent of those found in mammalian ERK5, ERK7 and ERK8. Expression analyses demonstrate that TbECK1 is constitutively expressed during the trypanosome life cycle at both RNA and protein level. In transgenic parasites we demonstrate that expression of a mutant of TbECK1 that lacks the C-terminal extension produces a slow growth phenotype, associated with the appearance of cells with aberrant karyotypes. Using this as an assay we further demonstrate that the phenotype is dependent upon the potential for catalytic activity of TbECK1 and on the integrity of at least one of the phosphorylable amino acids in its phosphorylation lip. C-terminal extensions are a common feature of kinetoplastid protein kinases. Our results demonstrate for the first time that this domain has a regulatory function.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/enzimología , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Ciclo Celular/fisiología , Proliferación Celular , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/genética , Quinasas MAP Reguladas por Señal Extracelular/química , Quinasas MAP Reguladas por Señal Extracelular/genética , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Fenotipo , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Alineación de Secuencia , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/fisiologíaRESUMEN
Phosphoenolpyruvate (PEP) mutase catalyzes the conversion of phosphoenolpyruvate to phosphonopyruvate, the initial step in the formation of many naturally occurring phosphonate compounds. The phosphonate compound 2-aminoethylphosphonate is present as a component of complex carbohydrates on the surface membrane of many trypanosomatids including glycosylinositolphospholipids of Trypanosoma cruzi. Using partial sequence information from the T. cruzi genome project we have isolated a full-length gene with significant homology to PEP mutase from the free-living protozoan Tetrahymena pyriformis and the edible mussel Mytilus edulis. Recombinant expression in Escherichia coli confirms that it encodes a functional PEP mutase with a Km apparent of 8 microM for phosphonopyruvate and a kcat of 12 s-1. The native enzyme is a homotetramer with an absolute requirement for divalent metal ions and displays negative cooperativity for Mg2+ (S0.5 0.4 microM; n = 0.46). Immunofluorescence and sub-cellular fractionation indicates that PEP mutase has a dual localization in the cell. Further evidence to support this was obtained by Western analysis of a partial sub-cellular fractionation of T. cruzi cells. Southern and Western analysis suggests that PEP mutase is unique to T. cruzi and is not present in the other medically important parasites, Trypanosoma brucei and Leishmania spp.