RESUMEN
FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is an imaging technique that allows 3D ultrastructural analysis of cells and tissues at the nanoscale. The acquired FIB-SEM data are highly noisy, which makes denoising an essential step prior to volume interpretation. Gaussian filtering is a standard method in the field because it is fast and straightforward. However, it tends to blur the biological features due to its linear nature that ignores the rapid changes of the structures throughout the volume. To address this issue, we have developed a new approach to structure-preserving noise reduction for FIB-SEM. It has abilities to locally adapt the filtering to the biological structures while taking advantage of the simplicity of Gaussian filtering. It uses the Optical Flow (OF) to estimate the variations of the structural features across the volume, so that they are compensated before the subsequent filtering with a Gaussian function. As demonstrated qualitatively and objectively with datasets from different samples and acquired under different conditions, our denoising approach outperforms the standard Gaussian filtering and is competitive with state-of-the-art methods in terms of noise reduction and preservation of the sharpness of the structures.
RESUMEN
BACKGROUND AND OBJECTIVE: Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) allows three-dimensional ultrastructural analysis of cells and tissues at the nanoscale. The technique iteratively removes a section of the sample with a FIB and takes an SEM image from the exposed surface. The section thickness is usually higher than the image pixel size to reduce acquisition time, thus resulting in anisotropic resolution. In this work, we explore novel interpolation methods along the sectioning direction to produce isotropic resolution and facilitate proper interpretation of the FIB-SEM 3D volumes. METHODS: Classical interpolation methods are usually applied in this context under the assumption that the changes through successive images are relatively smooth. However, the actual 3D arrangement of the structures in the sample may cause significant changes in the biological features between consecutive images of the FIB-SEM stacks. We have developed a novel interpolation strategy that accounts for this variation by using the Optical Flow (OF) to estimate it. As an intermediate stage, OF-compensated images are produced by aligning the spatial regions of the biological structures. Interpolated images are then generated from these OF-compensated images. The final isotropic stack is assembled by interleaving the interpolated images with the original images of the anisotropic stack. RESULTS: OF-driven and classical interpolation methods were compared using an objective assessment based on Pearson Correlation Coefficient (PCC) and a qualitative evaluation based on visual results, using public datasets and representative anisotropy conditions. The objective assessment demonstrated that the OF-driven interpolation always yields higher PCC values, with interpolated images closer to the ground truth. The qualitative evaluation corroborated those results and confirmed that classical interpolation may blur areas with substantial changes between consecutive images whereas OF-driven interpolation provides sharpness. CONCLUSIONS: We have developed an OF-driven interpolation approach to generating FIB-SEM stacks with isotropic resolution from experimental anisotropic data. It adapts to the rapid variation of the biological structures observed through the images of the FIB-SEM stack. Our approach outperforms classical interpolation and manages to produce sharp interpolated views in cases where there are significant changes between consecutive experimental images.
Asunto(s)
Flujo Optico , Anisotropía , Microscopía Electrónica de RastreoRESUMEN
Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine transferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV) isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified, suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.
Asunto(s)
Arabidopsis/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Virus Eruptivo de la Ciruela/fisiología , Acilación , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/virología , Cápside/metabolismo , Regulación Viral de la Expresión Génica , Genoma Viral , Datos de Secuencia Molecular , Mutación , Enfermedades de las Plantas/virología , Virus Eruptivo de la Ciruela/genética , Replicación ViralRESUMEN
A new plum pox potyvirus (PPV)-based vector has been constructed for the expression of full-length individual foreign proteins. The foreign sequences are cloned between the NIb replicase and capsid protein (CP) cistrons. The heterologous protein is split from the rest of the potyviral polyprotein by cleavage at the site that originally separated the NIb and CP proteins and at an additional NIa protease recognition site engineered at its amino-terminal end. This vector (PPV-NK) has been used to clone different genes, engendering stable chimeras with practical applications. We have constructed a chimera expressing high levels of jellyfish green fluorescent protein, which can be very useful for the study of PPV molecular biology. The VP60 structural protein of rabbit hemorrhagic disease virus (RHDV) was also successfully expressed by making use of the PPV-NK vector. Inoculation of extracts from VP60-expressing plants induced a remarkable immune response against RHDV in rabbits, its natural host. Moreover, these animals were protected against a lethal challenge with RHDV.
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Infecciones por Caliciviridae/prevención & control , Vectores Genéticos , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Nicotiana/genética , Plantas Tóxicas , Virus Eruptivo de la Ciruela , Proteínas Estructurales Virales/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Secuencia de Bases , ADN , Femenino , Expresión Génica , Genes Reporteros , Ingeniería Genética , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Proteínas Fluorescentes Verdes , Virus de la Enfermedad Hemorrágica del Conejo/genética , Proteínas Luminiscentes/genética , Datos de Secuencia Molecular , Virus Eruptivo de la Ciruela/genética , Virus Eruptivo de la Ciruela/inmunología , Conejos , Vacunación/métodos , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/genéticaRESUMEN
Plum pox potyvirus (PPV), the causal agent of a devastating disease that affects stone fruit trees, is becoming a target of intense studies intended both to fight against viral infection and to develop practical applications based on the current knowledge of potyvirus molecular biology. This review focuses on biotechnological aspects related to PPV, such as novel diagnostic techniques that facilitate detection and typing of virus isolates, strategies to implement pathogen-derived resistance through plant transformation, the potential use of genetic elements derived from the virus, and the recent development of PPV-based expression vectors.
Asunto(s)
Vectores Genéticos , Enfermedades de las Plantas/virología , Virus Eruptivo de la Ciruela/genética , Biotecnología/métodos , Elementos de Facilitación Genéticos , Enfermedades de las Plantas/estadística & datos numéricos , Plantas Modificadas Genéticamente/virología , Virus Eruptivo de la Ciruela/inmunología , Replicación ViralRESUMEN
The development of an antigen presentation system based on the plum pox potyvirus (PPV) is here described. The amino-terminal part of PPV capsid protein was chosen as the site for expression of foreign antigenic peptides. Modifications in this site were engineered to avoid the capability of natural transmission by aphids of this PPV vector. As a first practical attempt, different forms of an antigenic peptide (single and tandem repetition) from the VP2 capsid protein of canine parvovirus (CPV) were expressed. Both chimeras are able to infect Nicotiana clevelandii plants with similar characteristics to wild-type virus and remain genetically stable after several plant passages. The antigenicity of purified chimeric virions was demonstrated, proving the suitability of this system for diagnostic purposes. Moreover, mice and rabbits immunized with chimeric virions developed CPV-specific antibodies, which showed neutralizing activity.