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1.
Nature ; 578(7794): 273-277, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-32025029

RESUMEN

Synucleinopathies are neurodegenerative diseases that are associated with the misfolding and aggregation of α-synuclein, including Parkinson's disease, dementia with Lewy bodies and multiple system atrophy1. Clinically, it is challenging to differentiate Parkinson's disease and multiple system atrophy, especially at the early stages of disease2. Aggregates of α-synuclein in distinct synucleinopathies have been proposed to represent different conformational strains of α-synuclein that can self-propagate and spread from cell to cell3-6. Protein misfolding cyclic amplification (PMCA) is a technique that has previously been used to detect α-synuclein aggregates in samples of cerebrospinal fluid with high sensitivity and specificity7,8. Here we show that the α-synuclein-PMCA assay can discriminate between samples of cerebrospinal fluid from patients diagnosed with Parkinson's disease and samples from patients with multiple system atrophy, with an overall sensitivity of 95.4%. We used a combination of biochemical, biophysical and biological methods to analyse the product of α-synuclein-PMCA, and found that the characteristics of the α-synuclein aggregates in the cerebrospinal fluid could be used to readily distinguish between Parkinson's disease and multiple system atrophy. We also found that the properties of aggregates that were amplified from the cerebrospinal fluid were similar to those of aggregates that were amplified from the brain. These findings suggest that α-synuclein aggregates that are associated with Parkinson's disease and multiple system atrophy correspond to different conformational strains of α-synuclein, which can be amplified and detected by α-synuclein-PMCA. Our results may help to improve our understanding of the mechanism of α-synuclein misfolding and the structures of the aggregates that are implicated in different synucleinopathies, and may also enable the development of a biochemical assay to discriminate between Parkinson's disease and multiple system atrophy.


Asunto(s)
Atrofia de Múltiples Sistemas/diagnóstico , Enfermedad de Parkinson/diagnóstico , alfa-Sinucleína/líquido cefalorraquídeo , alfa-Sinucleína/química , Amiloide/química , Química Encefálica , Dicroismo Circular , Endopeptidasa K/metabolismo , Humanos , Atrofia de Múltiples Sistemas/líquido cefalorraquídeo , Enfermedad de Parkinson/líquido cefalorraquídeo , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , alfa-Sinucleína/clasificación , alfa-Sinucleína/toxicidad
2.
Proc Natl Acad Sci U S A ; 116(36): 18041-18049, 2019 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-31427528

RESUMEN

Assembly of pili on the gram-positive bacterial cell wall involves 2 conserved transpeptidase enzymes named sortases: One for polymerization of pilin subunits and another for anchoring pili to peptidoglycan. How this machine controls pilus length and whether pilus length is critical for cell-to-cell interactions remain unknown. We report here in Actinomyces oris, a key colonizer in the development of oral biofilms, that genetic disruption of its housekeeping sortase SrtA generates exceedingly long pili, catalyzed by its pilus-specific sortase SrtC2 that possesses both pilus polymerization and cell wall anchoring functions. Remarkably, the srtA-deficient mutant fails to mediate interspecies interactions, or coaggregation, even though the coaggregation factor CafA is present at the pilus tip. Increasing ectopic expression of srtA in the mutant progressively shortens pilus length and restores coaggregation accordingly, while elevated levels of shaft pilins and SrtC2 produce long pili and block coaggregation by SrtA+ bacteria. With structural studies, we uncovered 2 key structural elements in SrtA that partake in recognition of pilin substrates and regulate pilus length by inducing the capture and transfer of pilus polymers to the cell wall. Evidently, coaggregation requires proper positioning of the tip adhesin CafA via modulation of pilus length by the housekeeping sortase SrtA.


Asunto(s)
Actinomyces , Adhesinas Bacterianas , Aminoaciltransferasas , Proteínas Bacterianas , Cisteína Endopeptidasas , Fimbrias Bacterianas , Actinomyces/química , Actinomyces/genética , Actinomyces/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Fimbrias Bacterianas/química , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo
3.
Br J Nutr ; 126(3): 321-336, 2021 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32718370

RESUMEN

In the current research, a 60-d experiment was conducted with the purpose of exploring the impacts of methionine (Met) on growth performance, muscle nutritive deposition, muscle fibre growth and type I collagen synthesis as well as the related signalling pathway. Six diets (iso-nitrogenous) differing in Met concentrations (2·54, 4·85, 7·43, 10·12, 12·40 and 15·11 g/kg diets) were fed to 540 grass carp (178·47 (SD 0·36) g). Results showed (P < 0·05) that compared with Met deficiency, optimal level of dietary Met (1) increased feed intake, feed efficiency, specific growth rate and percentage weight gain (PWG); (2) increased fish muscle protein, lipid and free amino acid contents and improved fish muscle fatty acid profile as well as increased protein content in part associated with the target of rapamycin complex 1 (TORC1)/S6K1 signalling pathway; (3) increased the frequency distribution of muscle fibre with >50 µm of diameter; (4) increased type I collagen synthesis partly related to the transforming growth factor-ß1/Smads and CK2/TORC1 signalling pathways. In conclusion, dietary Met improved muscle growth, which might be due to the regulation of muscle nutritive deposition, muscle fibre growth and type I collagen synthesis-related signal molecules. Finally, according to PWG and muscle collagen content, the Met requirements for on-growing grass carp (178-626 g) were estimated to be 9·56 g/kg diet (33·26 g/kg protein of diet) and 9·28 g/kg diet (32·29 g/kg of dietary protein), respectively.


Asunto(s)
Carpas , Colágeno Tipo I/biosíntesis , Metionina/administración & dosificación , Fibras Musculares Esqueléticas/fisiología , Alimentación Animal/análisis , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Carpas/crecimiento & desarrollo , Dieta/veterinaria , Suplementos Dietéticos , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal
4.
Fish Shellfish Immunol ; 97: 216-234, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31857225

RESUMEN

The aim of this study was to investigate the effects of dietary biotin deficiency on the growth performance and immune function of the head kidney, spleen and skin in on-growing grass carp (Ctenopharyngodon idella). A total of 540 on-growing grass carp (117.11 ± 0.48 g) were fed six diets containing increasing levels of biotin (0.012, 0.110, 0.214, 0.311, 0.427 and 0.518 mg/kg diet) for 70 days. Subsequently, a challenge experiment was performed by infecting them with Aeromonas hydrophila for six days. Our results showed that compared with the appropriate biotin level, (1) biotin deficiency (0.012 mg/kg diet) reduced the activities of lysozyme (LZ) and acid phosphatase (ACP), decreased the contents of complement 3 (C3), C4 and immunoglobulin M (IgM), as well as reduced the mRNA levels of antimicrobial peptides in the head kidney, spleen and skin of on-growing grass carp; (2) biotin deficiency reduced the mRNA levels of anti-microbial substances: liver-expressed antimicrobial peptide (LEAP) -2A, LEAP-2B, hepcidin, ß-defensin-1 and mucin 2 in the head kidney, spleen and skin of on-growing grass carp; (3) biotin deficiency increased the mRNA levels of pro-inflammatory cytokines interleukin 1ß (IL-1ß), IL-6, IL-8, IL-12p40, IL-15, IL-17D, tumour necrosis factor α (TNF-α) and interferon γ2 (IFN-γ2) partially in association with nuclear factor-kappa B (NF-κB) signalling and reduced anti-inflammatory IL-4/13A, IL-10, IL-11 and transforming growth factor ß1 (TGF-ß1) mRNA levels partially in association with target of rapamycin (TOR) signalling in the head kidney, spleen and skin of on-growing grass carp. Interestingly, biotin deficiency had no effect on the expression of IL-12p35, IL-4/13B, TGF-ß2, 4E-BP1 (skin only) or IKKα in the head kidney, spleen and skin of on-growing grass carp. In conclusion, the results indicated that biotin deficiency impaired the immune function of the head kidney, spleen and skin in fish. Finally, based on the percent weight gain (PWG), the ability to prevent skin haemorrhages and lesions, the LZ activity in the head kidney and the C4 content in the spleen, the optimal dietary biotin levels for on-growing grass carp (117-534 g) were estimated as 0.210, 0.230, 0.245 and 0.238 mg/kg diet, respectively.


Asunto(s)
Deficiencia de Biotinidasa/veterinaria , Carpas , Enfermedades de los Peces/inmunología , Riñón Cefálico/inmunología , Inmunidad Innata/efectos de los fármacos , Piel/inmunología , Bazo/inmunología , Aeromonas hydrophila/fisiología , Alimentación Animal/análisis , Animales , Deficiencia de Biotinidasa/inmunología , Carpas/crecimiento & desarrollo , Dieta/veterinaria , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria
5.
Nature ; 502(7473): 707-10, 2013 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-24107993

RESUMEN

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.


Asunto(s)
Bacteriófagos/crecimiento & desarrollo , Bacteriófagos/ultraestructura , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Synechococcus/ultraestructura , Synechococcus/virología , Ensamble de Virus , Organismos Acuáticos/citología , Organismos Acuáticos/ultraestructura , Organismos Acuáticos/virología , Modelos Biológicos , Synechococcus/citología
6.
PLoS Biol ; 12(12): e1002024, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25514469

RESUMEN

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.


Asunto(s)
Bacteriófago PRD1/genética , Bacteriófago PRD1/ultraestructura , ADN Viral/genética , ADN/genética , Genoma Viral , Modelos Moleculares , Ensamble de Virus/genética , Cápside/química , Microscopía por Crioelectrón , ADN Viral/ultraestructura , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana , Membranas , Mutación/genética , Proteínas Virales , Virión/genética , Virión/ultraestructura
7.
J Virol ; 89(1): 676-87, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25355879

RESUMEN

UNLABELLED: A total of 2,691 mosquitoes representing 17 species was collected from eight locations in southwest Cameroon and screened for pathogenic viruses. Ten isolates of a novel reovirus (genus Dinovernavirus) were detected by culturing mosquito pools on Aedes albopictus (C6/36) cell cultures. A virus that caused overt cytopathic effects was isolated, but it did not infect vertebrate cells or produce detectable disease in infant mice after intracerebral inoculation. The virus, tentatively designated Fako virus (FAKV), represents the first 9-segment, double-stranded RNA (dsRNA) virus to be isolated in nature. FAKV appears to have a broad mosquito host range, and its detection in male specimens suggests mosquito-to-mosquito transmission in nature. The structure of the T=1 FAKV virion, determined to subnanometer resolution by cryoelectron microscopy (cryo-EM), showed only four proteins per icosahedral asymmetric unit: a dimer of the major capsid protein, one turret protein, and one clamp protein. While all other turreted reoviruses of known structures have at least two copies of the clamp protein per asymmetric unit, FAKV's clamp protein bound at only one conformer of the major capsid protein. The FAKV capsid architecture and genome organization represent the most simplified reovirus described to date, and phylogenetic analysis suggests that it arose from a more complex ancestor by serial loss-of-function events. IMPORTANCE: We describe the detection, genetic, phenotypic, and structural characteristics of a novel Dinovernavirus species isolated from mosquitoes collected in Cameroon. The virus, tentatively designated Fako virus (FAKV), is related to both single-shelled and partially double-shelled viruses. The only other described virus in this genus was isolated from cultured mosquito cells. It was previously unclear whether the phenotypic characteristics of that virus were reflective of this genus in nature or were altered during serial passaging in the chronically infected cell line. FAKV is a naturally occurring single-shelled reovirus with a unique virion architecture that lacks several key structural elements thought to stabilize a single-shelled reovirus virion, suggesting what may be the minimal number of proteins needed to form a viable reovirus particle. FAKV evolved from more complex ancestors by losing a genome segment and several virion proteins.


Asunto(s)
Culicidae/virología , Genoma Viral , Reoviridae/genética , Reoviridae/aislamiento & purificación , Animales , Camerún , Línea Celular , Análisis por Conglomerados , Microscopía por Crioelectrón , Efecto Citopatogénico Viral , Evolución Molecular , Especificidad del Huésped , Sustancias Macromoleculares/ultraestructura , Masculino , Ratones , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Reoviridae/fisiología , Reoviridae/ultraestructura , Análisis de Secuencia de ADN , Proteínas Estructurales Virales/ultraestructura , Virión/ultraestructura , Cultivo de Virus
8.
EMBO J ; 30(18): 3854-63, 2011 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-21829169

RESUMEN

Venezuelan equine encephalitis virus (VEEV), a member of the membrane-containing Alphavirus genus, is a human and equine pathogen, and has been developed as a biological weapon. Using electron cryo-microscopy (cryo-EM), we determined the structure of an attenuated vaccine strain, TC-83, of VEEV to 4.4 Å resolution. Our density map clearly resolves regions (including E1, E2 transmembrane helices and cytoplasmic tails) that were missing in the crystal structures of domains of alphavirus subunits. These new features are implicated in the fusion, assembly and budding processes of alphaviruses. Furthermore, our map reveals the unexpected E3 protein, which is cleaved and generally thought to be absent in the mature VEEV. Our structural results suggest a mechanism for the initial stage of nucleocapsid core formation, and shed light on the virulence attenuation, host recognition and neutralizing activities of VEEV and other alphavirus pathogens.


Asunto(s)
Virus de la Encefalitis Equina Venezolana/ultraestructura , Animales , Microscopía por Crioelectrón , Caballos , Modelos Moleculares , Proteínas Virales/ultraestructura , Vacunas Virales , Virión/ultraestructura , Virulencia
9.
J Virol ; 85(18): 9327-33, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21752915

RESUMEN

Barmah Forest virus (BFV) is a mosquito-borne alphavirus that infects humans. A 6-Å-resolution cryo-electron microscopy three-dimensional structure of BFV exhibits a typical alphavirus organization, with RNA-containing nucleocapsid surrounded by a bilipid membrane anchored with the surface proteins E1 and E2. The map allows details of the transmembrane regions of E1 and E2 to be seen. The C-terminal end of the E2 transmembrane helix binds to the capsid protein. Following the E2 transmembrane helix, a short α-helical endodomain lies on the inner surface of the lipid envelope. The E2 endodomain interacts with E1 transmembrane helix from a neighboring E1-E2 trimeric spike, thereby acting as a spacer and a linker between spikes. In agreement with previous mutagenesis studies, the endodomain plays an important role in recruiting other E1-E2 spikes to the budding site during virus assembly. The E2 endodomain may thus serve as a target for antiviral drug design.


Asunto(s)
Alphavirus/ultraestructura , Sustancias Macromoleculares/ultraestructura , Proteínas Virales/ultraestructura , Virión/ultraestructura , Animales , Microscopía por Crioelectrón , Humanos , Imagenología Tridimensional , Modelos Moleculares , Nucleocápside/ultraestructura
10.
Adv Exp Med Biol ; 726: 49-90, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22297510

RESUMEN

The past few decades have seen tremendous advances in single-particle electron -cryo-microscopy (cryo-EM). The field has matured to the point that near-atomic resolution density maps can be generated for icosahedral viruses without the need for crystallization. In parallel, substantial progress has been made in determining the structures of nonicosahedrally arranged proteins in viruses by employing either single-particle cryo-EM or cryo-electron tomography (cryo-ET). Implicit in this course have been the availability of a new generation of electron cryo-microscopes and the development of the computational tools that are essential for generating these maps and models. This methodology has enabled structural biologists to analyze structures in increasing detail for virus particles that are in different morphogenetic states. Furthermore, electron imaging of frozen, hydrated cells, in the process of being infected by viruses, has also opened up a new avenue for studying virus structures "in situ". Here we present the common techniques used to acquire and process cryo-EM and cryo-ET data and discuss their implications for structural virology both now and in the future.


Asunto(s)
Microscopía por Crioelectrón/métodos , Tomografía/métodos , Virus/ultraestructura , Animales , Cápside/ultraestructura , Humanos , Imagenología Tridimensional/métodos , Modelos Moleculares , Conformación Proteica , Proteínas Virales/química
11.
Nat Commun ; 13(1): 379, 2022 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-35046412

RESUMEN

Bacterial type IV secretion systems (T4SSs) are largely responsible for the proliferation of multi-drug resistance. We solved the structure of the outer-membrane core complex (OMCCF) of a T4SS encoded by a conjugative F plasmid at <3.0 Å resolution by cryoelectron microscopy. The OMCCF consists of a 13-fold symmetrical outer ring complex (ORC) built from 26 copies of TraK and TraV C-terminal domains, and a 17-fold symmetrical central cone (CC) composed of 17 copies of TraB ß-barrels. Domains of TraV and TraB also bind the CC and ORC substructures, establishing that these proteins undergo an intraprotein symmetry alteration to accommodate the C13:C17 symmetry mismatch. We present evidence that other pED208-encoded factors stabilize the C13:C17 architecture and define the importance of TraK, TraV and TraB domains to T4SSF function. This work identifies OMCCF structural motifs of proposed importance for structural transitions associated with F plasmid dissemination and F pilus biogenesis.


Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Factor F/metabolismo , Sistemas de Secreción Tipo IV/química , Membrana Celular/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Sistemas de Secreción Tipo IV/ultraestructura
12.
Front Immunol ; 12: 742107, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34733280

RESUMEN

The objective of this study was to evaluate the efficacy of dietary Mannan oligosaccharides (MOS) supplementation on skin barrier function and the mechanism of on-growing grass carp (Ctenopharyngodon idella). Five hundred forty grass carp were fed for 60 days from the growing stage with six different levels of MOS diets (0, 200, 400, 600, 800, and 1,000 mg kg-1). At the end of the growth trial, the 14-day Aeromonas hydrophila challenge experiment has proceeded. The obtained data indicate that MOS could (1) decline skin lesion morbidity after being challenged by the pathogenic bacteria; (2) maintain physical barrier function via improving antioxidant ability, inhibiting excessive apoptosis, and strengthening the tight junction between the epithelial cell and the related signaling pathway (Nrf2/Keap1, p38MAPK, and MLCK); and (3) regulate immune barrier function by modulating the production of antimicrobial compound and expression of involved cytokines and the related signaling pathway (TOR and NFκB). Finally, we concluded that MOS supplementation reinforced the disease resistance and protected the fish skin barrier function from Aeromonas hydrophila infection.


Asunto(s)
Carpas/inmunología , Enfermedades de los Peces/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Mananos/farmacología , Piel/inmunología , Aeromonas hydrophila , Alimentación Animal , Animales , Enfermedades de los Peces/microbiología , Prebióticos
13.
Toxicon ; 174: 32-42, 2020 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-31881237

RESUMEN

Deoxynivalenol (DON) is a common mycotoxin existed in animal feed, and lead to significant economic loss due to its negative impacts on animal growth performance and animal health. The gill is a primary mucosal immune organ in teleosts, and the structural integrity of the gill is closely relevant with fish healthy growth. Hence, this study assessed the influences of DON on the gill structural integrity of juvenile grass carp, Ctenopharyngodan idella (initial average weight 12.17 ± 0.01 g), when offered with six different diets which contained various content of DON (27, 318, 636, 922, 1243 and 1515 µg/kg diet) for 60 days. Our research firstly systematically elaborated that DON caused histopathological lesions, oxidative injury, reduction of antioxidant ability, apoptosis as well as damages of tight junctions in fish gills. Comparing these data to the control, we found that DON at dose of more than 318 µg/kg diet led to oxidative injury, apoptosis and disruption of tight junctions in fish gill, which were likely to be relevant with Nrf2, JNK and MLCK signalling pathways, respectively. It was worth noting that DON was not found to affect the gene expressions of Keap1b (rather than Keap1a), claudin-b, claudin-3c and claudin-15b (not claudin-15a) in fish gills. Furthermore, based on MDA and T-AOC activities in the gill, the maximum permissible levels of DON were evaluated to be 375.60 as well as 412.91 µg/kg diet in grass carp, respectively.


Asunto(s)
Branquias/efectos de los fármacos , Tricotecenos/toxicidad , Alimentación Animal , Animales , Antioxidantes , Apoptosis , Carpas , Dieta , Suplementos Dietéticos , Proteínas de Peces/metabolismo , Branquias/fisiología , Estrés Oxidativo/fisiología , Transducción de Señal , Uniones Estrechas/efectos de los fármacos
14.
J Struct Biol ; 165(1): 1-9, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18926912

RESUMEN

The JEOL Automated Data Acquisition System (JADAS) is a software system built for the latest generation of the JEOL Transmission Electron Microscopes. It is designed to partially or fully automate image acquisition for ice-embedded single particles under low dose conditions. Its built-in flexibility permits users to customize the order of various imaging operations. In this paper, we describe how JADAS is used to accurately locate and image suitable specimen areas on a grid of ice-embedded particles. We also demonstrate the utility of JADAS by imaging the epsilon 15 bacteriophage with the JEM3200FSC electron cryo-microscope, showing that sufficient images can be collected in a single 8h session to yield a subnanometer resolution structure which agrees with the previously determined structure.


Asunto(s)
Bacteriófagos/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Algoritmos , Automatización , Microscopía por Crioelectrón , Hielo , Procesamiento de Imagen Asistido por Computador/instrumentación
15.
J Struct Biol ; 163(1): 45-52, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18514542

RESUMEN

Bacteriorhodopsin and epsilon 15 bacteriophage were used as biological test specimens to evaluate the potential structural resolution with images captured from a 4k x 4k charge-coupled device (CCD) camera in a 300-kV electron cryomicroscope. The phase residuals computed from the bacteriorhodopsin CCD images taken at 84,000x effective magnification averaged 15.7 degrees out to 5.8-A resolution relative to Henderson's published values. Using a single-particle reconstruction technique, we obtained an 8.2-A icosahedral structure of epsilon 15 bacteriophage with the CCD images collected at an effective magnification of 56,000x. These results demonstrate that it is feasible to retrieve biological structures to a resolution close to 2/3 of the Nyquist frequency from the CCD images recorded in a 300-kV electron cryomicroscope at a moderately high but practically acceptable microscope magnification.


Asunto(s)
Microscopía por Crioelectrón/instrumentación , Bacteriófagos/ultraestructura , Bacteriorodopsinas/ultraestructura , Microscopía por Crioelectrón/normas , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/normas
17.
Phys Rev E Stat Nonlin Soft Matter Phys ; 66(6 Pt 2): 066703, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12513440

RESUMEN

A real space approach has been proposed to solve the x-ray phase problem formulated as a minimization problem. The cost function consists of two parts: one represents the usual crystallographical residual while the other enforces the probability distribution of the invariant phase triplets. Starting from a random real space structure, the atoms are moved one by one to gradually reduce the cost function (simulated annealing). In addition, the atoms are encouragd to preferentially sample the high density regions in space determined by an approximate density map which in turn is updated and modified by averaging and Fourier synthesis. Such a reduction of the configurational space has led to considerable improvement of the algorithm compared to an earlier version. Trial calculations for structures including hexadecaisoleucinomycin (HEXIL) and a collagenlike peptide (PPG) are presented.

18.
Bacteriophage ; 4(4): e961869, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-26713220

RESUMEN

Drug development has typically been a primary foundation of strategy for systematic, long-range management of pathogenic cells. However, drug development is limited in speed and flexibility when response is needed to changes in pathogenic cells, especially changes that produce drug-resistance. The high replication speed and high diversity of phages are potentially useful for increasing both response speed and response flexibility when changes occur in either drug resistance or other aspects of pathogenic cells. We present strategy, with some empirical details, for (1) using modern molecular biology and biophysics to access these advantages during the phage therapy of bacterial infections, and (2) initiating use of phage capsid-based drug delivery vehicles (DDVs) with procedures that potentially overcome both drug resistance and other present limitations in the use of DDVs for the therapy of neoplasms. The discussion of phage therapy includes (a) historical considerations, (b) changes that appear to be needed in clinical tests if use of phage therapy is to be expanded, (c) recent work on novel phages and its potential use for expanding the capabilities of phage therapy and (d) an outline for a strategy that encompasses both theory and practice for expanding the applications of phage therapy. The discussion of DDVs starts by reviewing current work on DDVs, including work on both liposomal and viral DDVs. The discussion concludes with some details of the potential use of permeability constrained phage capsids as DDVs.

19.
Nat Commun ; 5: 4808, 2014 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-25185801

RESUMEN

Advances in electron cryo-microscopy have enabled structure determination of macromolecules at near-atomic resolution. However, structure determination, even using de novo methods, remains susceptible to model bias and overfitting. Here we describe a complete workflow for data acquisition, image processing, all-atom modelling and validation of brome mosaic virus, an RNA virus. Data were collected with a direct electron detector in integrating mode and an exposure beyond the traditional radiation damage limit. The final density map has a resolution of 3.8 Å as assessed by two independent data sets and maps. We used the map to derive an all-atom model with a newly implemented real-space optimization protocol. The validity of the model was verified by its match with the density map and a previous model from X-ray crystallography, as well as the internal consistency of models from independent maps. This study demonstrates a practical approach to obtain a rigorously validated atomic resolution electron cryo-microscopy structure.


Asunto(s)
Bromovirus/ultraestructura , Microscopía por Crioelectrón/métodos , Electrones , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Microscopía por Crioelectrón/instrumentación , Cristalografía por Rayos X , Modelos Moleculares , Conformación Proteica
20.
Structure ; 18(8): 903-12, 2010 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-20696391

RESUMEN

Zernike phase contrast cryo-electron microscopy (ZPC-cryoEM) is an emerging technique that is capable of producing higher image contrast than conventional cryoEM. By combining this technique with advanced image processing methods, we achieved subnanometer resolution for two biological specimens: 2D bacteriorhodopsin crystal and epsilon15 bacteriophage. For an asymmetric reconstruction of epsilon15 bacteriophage, ZPC-cryoEM can reduce the required amount of data by a factor of approximately 3, compared with conventional cryoEM. The reconstruction was carried out to 13 A resolution without the need to correct the contrast transfer function. New structural features at the portal vertex of the epsilon15 bacteriophage are revealed in this reconstruction. Using ZPC cryo-electron tomography (ZPC-cryoET), a similar level of data reduction and higher resolution structures of epsilon15 bacteriophage can be obtained relative to conventional cryoET. These results show quantitatively the benefits of ZPC-cryoEM and ZPC-cryoET for structural determinations of macromolecular machines at nanometer and subnanometer resolutions.


Asunto(s)
Microscopía por Crioelectrón/métodos , Cristalografía/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía de Contraste de Fase/métodos , Modelos Moleculares , Bacteriófagos/ultraestructura , Bacteriorodopsinas/ultraestructura , Epsilonproteobacteria/virología
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