RESUMEN

Many-body Green's function perturbation theories, such as the GW and Bethe-Salpeter formalisms, are starting to be routinely applied to study charged and neutral electronic excitations in molecular organic systems relevant to applications in photovoltaics, photochemistry or biology. In parallel, density functional theory and its time-dependent extensions significantly progressed along the line of range-separated hybrid functionals within the generalized Kohn-Sham formalism designed to provide correct excitation energies. We give an overview and compare these approaches with examples drawn from the study of gas phase organic systems such as fullerenes, porphyrins, bacteriochlorophylls or nucleobases molecules. The perspectives and challenges that many-body perturbation theory is facing, such as the role of self-consistency, the calculation of forces and potential energy surfaces in the excited states, or the development of embedding techniques specific to the GW and Bethe-Salpeter equation formalisms, are outlined.

Asunto(s)

Bioquímica/métodos , Compuestos Orgánicos/química , Fotoquímica/métodos , Algoritmos , Bacterioclorofilas/química , Catálisis , Electrones , Fulerenos/química , Gases , Modelos Teóricos , Óptica y Fotónica , Porfirinas/química , Zinc/química

RESUMEN

We study within the many-body Green's function GW and Bethe-Salpeter formalisms the excitation energies of a paradigmatic model dipeptide, focusing on the four lowest-lying local and charge-transfer excitations. Our GW calculations are performed at the self-consistent level, updating first the quasiparticle energies, and further the single-particle wavefunctions within the static Coulomb-hole plus screened-exchange approximation to the GW self-energy operator. Important level crossings, as compared to the starting Kohn-Sham LDA spectrum, are identified. Our final Bethe-Salpeter singlet excitation energies are found to agree, within 0.07 eV, with CASPT2 reference data, except for one charge-transfer state where the discrepancy can be as large as 0.5 eV. Our results agree best with LC-BLYP and CAM-B3LYP calculations with enhanced long-range exchange, with a 0.1 eV mean absolute error. This has been achieved employing a parameter-free formalism applicable to metallic or insulating extended or finite systems.

Asunto(s)

Dipéptidos/química , Teoría Cuántica , Modelos Moleculares , Fenómenos Ópticos

RESUMEN

OBJECTIVES: In recent years, one of the foci of orthodontics has been on systems for the evaluation of treatment results and the tracking of tissue variations over time. This can be accomplished through analysing three-dimensional orthodontic images obtained before and after the treatments. Since complementary information is achieved by integrating multiple imaging modalities, cone beam CT (CBCT) and stereophotogrammetry technologies are used in this study to develop a method for tracking bone, teeth and facial soft-tissue variations over time. METHODS: We propose a two-phase procedure of multimodal (Phase 1) and multitemporal (Phase 2) registration which aligns images taken from the same patient by different imaging modalities and at different times. Extrinsic (for Phase 1) and intrinsic (for Phase 2) landmark-based registration methods are employed as an initiation for a robust iterative closest points algorithm. Since the mandible moves independently of the upper skull, the registration procedure is applied separately on the mandible and the upper skull. RESULTS: The results show that the signed error distributions of both mandible and skull registrations follow a mixture of two Gaussian distributions, corresponding to alignment errors (due to our method) and temporal change over time. CONCLUSIONS: We suggest that the large values among the total registration errors correspond to the temporal change resulting from (1) the effect of treatment (i.e. the orthodontic changes of teeth positions); (2) the biological changes such as teeth growth over time, especially for teenagers; and (3) the segmentation procedure and CBCT precision change over time.

Asunto(s)

Cefalometría/métodos , Tomografía Computarizada de Haz Cónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Fotogrametría/métodos , Adolescente , Algoritmos , Puntos Anatómicos de Referencia/diagnóstico por imagen , Cefalometría/estadística & datos numéricos , Niño , Tomografía Computarizada de Haz Cónico/estadística & datos numéricos , Huesos Faciales/crecimiento & desarrollo , Femenino , Marcadores Fiduciales , Estudios de Seguimiento , Humanos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Imagenología Tridimensional/estadística & datos numéricos , Masculino , Mandíbula/diagnóstico por imagen , Mandíbula/crecimiento & desarrollo , Desarrollo Maxilofacial/fisiología , Distribución Normal , Odontogénesis/fisiología , Ortodoncia Correctiva , Fotogrametría/estadística & datos numéricos , Cráneo/diagnóstico por imagen , Cráneo/crecimiento & desarrollo

RESUMEN

Lacosamide (Vimpat®) is an antiepileptic drug approved in the USA, Europe and several other countries as adjunctive therapy for partial-onset seizures. We report a simple HPLC method with UV detection for the quantification of lacosamide in human plasma. The method involves protein precipitation with methanol followed by chromatographic separation using an ACE® C18-AR column (2.1 mm × 150 mm, 3.0 µm) and mobile phases consisting of mixtures of ammonium formate buffer at pH 9 and acetonitrile. Briefly, 25 µl of internal standard and 300 µl of methanol are added to 100 µl of plasma. After vortexing and centrifugation, 70 µl of supernatant is transferred to an autosampler vial and 5.0 µl is injected. Calibration curves are linear in the range of 0.5 to 12.5 µg/ml. A validation was performed that consisted of the evaluation of accuracy and precision, specificity, limit of detection and carryover. Moreover, the possibility of using single-point calibration was evaluated and a crossvalidation between this method and an established LC-MS/MS method using pooled clinical study samples was performed. The method's sensitivity, simplicity and reliance on simpler HPLC equipment should allow for straightforward application in drug monitoring.

Asunto(s)

Acetamidas/sangre , Anticonvulsivantes/sangre , Cromatografía Líquida de Alta Presión/métodos , Espectrofotometría Ultravioleta/métodos , Monitoreo de Drogas/métodos , Femenino , Humanos , Lacosamida , Masculino , Espectrometría de Masas en Tándem/métodos

RESUMEN

This paper describes a new analysis method dedicated to the comparison of human gestures. The orientations and the positions of the gestures are first digitized using active 3D sensors and then compared to a 6-D template using curvature analysis. The proposed algorithm first starts by computing the invariant curvature of 3D position and orientation of a surgical tool using Frenet-Serret frames in 3D and quaternion space. The resulting curvature calculation is matched and compared to the template using a Dynamic Time Warping method. The proposed method is invariant to sensor position and orientation. An experimental study shows the efficiency of the new algorithm for an application in obstetrics, where the aim is to compare forceps blade placements between a senior medical doctor and a novice.

Asunto(s)

Algoritmos , Gestos , Obstetricia/métodos , Percepción Espacial/fisiología , Femenino , Humanos , Forceps Obstétrico , Embarazo

RESUMEN

Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection-binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios.

Asunto(s)

Bacteriófagos/metabolismo , ADN Viral/metabolismo , Modelos Biológicos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismo , Activación del Canal Iónico , Cinética , Unión Proteica , Factores de Tiempo

RESUMEN

Human adenoviruses (HAdV) are widely used for in vitro and in vivo gene transfer. Viral hepatotropism, inflammatory responses and neutralization by pre-existing antibodies (NAbs) are obstacles for clinical applications of HAdV vectors. Although the multifactorial events leading to innate HAdV toxicity are far from being elucidated, there is a consensus that the majority of intravenously injected-HAdV vectors is sequestered by Kuppfer cells, probably independently of coagulation factors. In this study, we show that the adenoviral-associated humoral and innate cytokine immune responses are significantly reduced when HAdV-5 vector carrying human bovine chimeric fibers (HAdV-5-F2/BAdV-4) is intravenously injected into mice. Fiber pseudotyping modified its interaction with blood coagulation factors, as FIX and FX no longer mediate the infection of liver cells by HAdV-5-F2/BAdV-4. As a consequence, at early time points post-infection, several cytokines and chemokines (IFN-gamma, IL-6, IP-10, MCP-1, RANTES and MP1beta) were found to be present at lower levels in the plasma of mice that had been intravenously injected with HAdV-5-F2/BAdV-4 compared with mice injected with the parental vector HAdV-5. Moreover, genetic modification of the fiber allowed HAdV-5-F2/BAdV-4 to partially escape neutralization by NAbs.

Asunto(s)

Adenoviridae/genética , Adenovirus Humanos/genética , Quimera , Hepatocitos/virología , Inmunidad Innata , Adenoviridae/inmunología , Adenoviridae/patogenicidad , Adenovirus Humanos/inmunología , Animales , Anticuerpos Antivirales , Factores de Coagulación Sanguínea/metabolismo , Bovinos , Línea Celular , Quimiocinas/análisis , Citocinas/análisis , Vectores Genéticos , Genoma Viral , Humanos , Inflamación/virología , Ratones , Transducción Genética

RESUMEN

This paper presents a method to evaluate medical gestures. The objective is to objectively assess a gesture carried out by novice doctors. The proposed method is based on the study of the curvature of the 3D gesture and provide a global performance index for one manipulation. The study of the number of peaks on the curvature indicates if the gesture is smooth or not. The application is the obstetric gestures linked to the forceps use but the method can be applied to different gestures without loss of generality. Seven residents carried out 30 forceps blade placements. The results clearly show a difference between the gestures carried out. This highlights the difficulty of the gesture according to the fetal head presentation.

Asunto(s)

Instrucción por Computador/métodos , Parto Obstétrico/instrumentación , Parto Obstétrico/métodos , Evaluación Educacional/métodos , Gestos , Modelos Biológicos , Competencia Profesional , Simulación por Computador , Diseño Asistido por Computadora , Forceps Obstétrico

RESUMEN

This paper focuses on the gesture analysis in order to compare two human gestures. The orientations and the positions of the gestures are both taken into account and the similarity rate between two gestures is calculated. In our case, the application is in obstetrics and the aim is to evaluate forceps blade placement. The method is based on the curvature analysis of the paths during the gesture. The 3-D position paths are expressed according to their cumulated chord length and the orientation paths in the quaternion unit space. These parameterizations lead to analyze data in space independently to time as requested by physicians. After filtering data in order to minimize sensor noises, the gestures are then compared by calculating the correlation between the position and the orientation curvatures of a novice gesture and an expert one. The results clearly show that novice skills in handling forceps increase in becoming smoother and closer to the reference placement. A childbirth simulator allows novices to acquire experience without any risks, however the training have to be completed with the extraction gesture evaluation and a compulsory training period in the delivery ward.

Asunto(s)

Capacitación en Servicio , Modelos Anatómicos , Obstetricia , Fenómenos Electromagnéticos , Femenino , Humanos , Embarazo , Instrumentos Quirúrgicos

RESUMEN

This paper presents a method to evaluate a gesture carried out by a resident obstetrician by comparing it to a gesture carried out by an expert obstetrician. The studied gesture is the forceps blade placement. Resident paths were recorded on a childbirth simulator while placing forceps blades instrumented with six degrees of freedom sensors. The path is characterized by the positions and the orientations. In this paper we particularly focus on the orientations. Forceps orientations are expressed in the quaternion unit space and the curvature of quaternion path is compared by correlation to a reference defined by an expert. Residents have been trained on a simulator and their gestures are evaluated by comparing their orientation path curvatures to reference path curvatures. Quantitative results confirm the qualitative analysis, residents become more similar to the reference while training on simulator.

Asunto(s)

Instrucción por Computador/instrumentación , Extracción Obstétrica/educación , Extracción Obstétrica/instrumentación , Imagenología Tridimensional/instrumentación , Parto , Cirugía Asistida por Computador/métodos , Instrucción por Computador/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Extracción Obstétrica/métodos , Femenino , Gestos , Humanos , Imagenología Tridimensional/métodos , Forceps Obstétrico , Obstetricia/educación , Obstetricia/instrumentación , Obstetricia/métodos , Embarazo , Riesgo , Instrumentos Quirúrgicos

RESUMEN

This paper presents a method to evaluate a gesture carried out by a resident obstetrician doctors by comparing it to a gesture carried out by an expert obstetrician doctors. The studied gesture is the forceps blade placement. Residents were recorded on a childbirth simulator while placing forceps blades. Their paths were compared in order to evaluate how similar they are to a reference path defined by an expert. The comparison method is developed with respect to expert requests: time independence and in considering the whole set of data and not only particular points. In order to respect these requests, the developed method lies on the correlation coefficient between the path curvatures. Residents have been trained on a simulator and their gestures were evaluated by comparing their path curvatures to reference path curvatures. Quantitative results confirm the qualitative analysis, residents become more similar to the reference while training on simulator.

Asunto(s)

Instrucción por Computador/instrumentación , Extracción Obstétrica/educación , Extracción Obstétrica/instrumentación , Gestos , Imagenología Tridimensional/instrumentación , Forceps Obstétrico , Cirugía Asistida por Computador/métodos , Instrucción por Computador/métodos , Diseño de Equipo , Análisis de Falla de Equipo , Extracción Obstétrica/métodos , Imagenología Tridimensional/métodos

RESUMEN

In order to use adenovirus (Ad) type 5 (Ad5) for cancer gene therapy, Ad needs to be de-targeted from its native receptors and re-targeted to a tumor antigen. A limiting factor for this has been to find a ligand that (i) binds a relevant target, (ii) is able to fold correctly in the reducing environment of the cytoplasm and (iii) when incorporated at an optimal position on the virion results in a virus with a low physical particle to plaque-forming units ratio to diminish the viral load to be administered to a future patient. Here, we present a solution to these problems by producing a genetically re-targeted Ad with a tandem repeat of the HER2/neu reactive Affibody molecule (ZH) in the HI-loop of a Coxsackie B virus and Ad receptor (CAR) binding ablated fiber genetically modified to contain sequences for flexible linkers between the ZH and the knob sequences. ZH is an Affibody molecule specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) that is overexpressed in inter alia breast and ovarian carcinomas. The virus presented here exhibits near wild-type growth characteristics, infects cells via HER2/neu instead of CAR and represents an important step toward the development of genetically re-targeted adenoviruses with clinical relevance.

Asunto(s)

Adenoviridae/genética , Antígenos de Neoplasias/genética , Terapia Genética/métodos , Vectores Genéticos/genética , Proteínas Recombinantes de Fusión/genética , Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/terapia , Femenino , Humanos , Ligandos , Neoplasias Ováricas/terapia , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales Cultivadas

RESUMEN

In this study, a prototype Adenovirus type 5 (Ad5) vector deleted of the fiber knob domain and carrying an Affibody molecule as the targeting ligand showed decreased susceptibility to human pre-existing antibodies. This vector, Ad5/R7-Z(taq)Z(taq), has short fibers carrying seven shaft repeats, a non-native trimerization signal and an affibody molecule (Z(taq)) reactive to Taq polymerase. Ad5/R7-Z(taq)Z(taq) could be specifically targeted to 293 cells stably expressing membrane-bound anti-Z(taq) idiotypic affibody called Z(ztaq) (293Z(ztaq)). Sera from 50 blood donors were analyzed for neutralization activity (NA) against the parental Ad5/Fiwt vector and knobless Ad5/R7-Z(taq)Z(taq) on 293Z(ztaq) cells. Twenty-three sera had NA titers (> or =1:64) against Ad5/Fiwt (46%) and only two against Ad5/R7-Z(taq)Z(taq) (4%). Characterization of sera with NA titers showed that the knob domain is one of the targets of the antibodies. Neutralization assays using sera pre-adsorbed on knob and hexon proteins showed that the NA of the sera was carried mainly by anti-knob and anti-hexon antibodies, but in certain sera the anti-hexon antibodies represent the major population of the neutralizing antibodies (NAbs). Our results suggested that a combination of knob deletion and hexon switching could be an effective strategy for Ad vectors to better evade the anti-Ad NAbs.

Asunto(s)

Adenovirus Humanos/genética , Anticuerpos Antivirales/inmunología , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Adenovirus Humanos/inmunología , Anticuerpos Antivirales/sangre , Antígenos Virales/genética , Proteínas de la Cápside/genética , Eliminación de Gen , Ingeniería Genética , Humanos , Pruebas de Neutralización , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología

RESUMEN

Evolutionary relationships between viruses may be obscure by protein sequence but unmasked by structure. Analysis of bacteriophage T5 by cryo-electron microscopy and protein sequence analysis reveals analogies with HK97 and T4 that suggest a mosaic of such connections. The T5 capsid is consistent with the HK97 capsid protein fold but has a different geometry, incorporating three additional hexamers on each icosahedral facet. Similarly to HK97, the T5 major capsid protein has an N-terminal extension, or Delta-domain that is missing in the mature capsid, and by analogy with HK97, may function as an assembly or scaffold domain. This Delta-domain is predicted to be largely coiled-coil, as for that of HK97, but is approximately 70% longer correlating with the larger capsid. Thus, capsid architecture appears likely to be specified by the Delta-domain. Unlike HK97, the T5 capsid binds a decoration protein in the center of each hexamer similarly to the "hoc" protein of phage T4, suggesting a common role for these molecules. The tail-tube has unusual trimeric symmetry that may aid in the unique two-stage DNA-ejection process, and joins the tail-tip at a disk where tail fibers attach. This intriguing mix of characteristics embodied by phage T5 offers insights into virus assembly, subunit function, and the evolutionary connections between related viruses.

Asunto(s)

Bacteriófago T4/química , Bacteriófago T4/ultraestructura , Proteínas de la Cápside/química , Evolución Molecular , Siphoviridae/química , Siphoviridae/ultraestructura , Secuencia de Aminoácidos , Cápside/química , Microscopía por Crioelectrón , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

RESUMEN

Ucb-35440-3 is a new drug entity under investigation at UCB S.A. Due to its physicochemical characteristics, the drug, a poorly water-soluble weak base, shows poor solubility and dissolution characteristics. In rat, the low oral bioavailability (F < 10%) is largely due to poor absorption. In order to enhance the solubility and dissolution characteristics, formulation of ucb-35440-3 as nanocrystals has been achieved in this study. Nanoparticles were prepared using high pressure homogenization and were characterized in terms of size and morphology. In vitro dissolution characteristics were investigated and compared to the un-milled drug in order to verify the theoretical hypothesis on the benefit of increased surface area. In vivo pharmacokinetic evaluation of ucb-35440-3 nanoparticles was also carried out on rats. Crystalline state evaluation before and following particle size reduction was conducted through polarized light microscopy and PXRD to denote any possible transformation to an amorphous state during the homogenization process. Drug chemical stability was also assessed following homogenization. The dissolution rate increased significantly at pH 3.0, 5.0 and 6.5 for ucb-35440-3 nanoparticles. However, the pharmacokinetic profile obtained yielded lower systemic exposure than the un-milled compound (in fed state), this although being thought to be the consequence of the drug and formulation characteristics.

Asunto(s)

Antiasmáticos/farmacocinética , Benzamidas/farmacocinética , Nanopartículas , Piperazinas/farmacocinética , Administración Oral , Animales , Antiasmáticos/administración & dosificación , Antiasmáticos/sangre , Antiasmáticos/química , Benzamidas/administración & dosificación , Benzamidas/sangre , Benzamidas/química , Disponibilidad Biológica , Química Farmacéutica , Cristalización , Estabilidad de Medicamentos , Excipientes/química , Concentración de Iones de Hidrógeno , Derivados de la Hipromelosa , Masculino , Metilcelulosa/análogos & derivados , Metilcelulosa/química , Tamaño de la Partícula , Piperazinas/administración & dosificación , Piperazinas/sangre , Piperazinas/química , Presión , Ratas , Ratas Wistar , Solubilidad , Tecnología Farmacéutica , Factores de Tiempo

RESUMEN

For the next decade, COPD will become the third cause of mortality in the world. COPD is mainly due to cigarette smoking and presents different levels of severity according to people, probably linked to environmental and genetic factors, which are not well documented. Recent publications pointed out bacterial bronchial colonization and exacerbations of infectious origin as worsening factors through a pro-inflammatory effect and oxidative stress. This should lead to a comprehensive review of anti-infectious prevention tools and to discuss the role of prophylactic antibiotherapy and antioxidants.

Asunto(s)

Control de Enfermedades Transmisibles , Infecciones/etiología , Estrés Oxidativo/fisiología , Enfermedad Pulmonar Obstructiva Crónica/fisiopatología , Ambiente , Humanos , Enfermedad Pulmonar Obstructiva Crónica/complicaciones , Enfermedad Pulmonar Obstructiva Crónica/epidemiología , Fumar/efectos adversos

RESUMEN

Oxidative stress is a frequent mechanism involved in the pathogenesis of bronchopulmonary disease. The cause can be exogenous, in particular related to to atmospheric pollution and tobacco smoke, or endogenous, related to mobilization of inflammatory cells (macrophages and polymorphonuclear neutrophils). In this general review, we present work demonstrating this oxidative stress and activation of inflammatory cells. We discuss the effect of oxidative stress on the bronchial tree and the need to maintain an adequate balance between oxidants and anti-oxidants. This reviews focuses on experimental studies proving the anti-oxidant effect of NAC on glutathione synthesis and on different pharmacological models. We then discuss human trials, initially experimental then in different bronchopulmonary pathologies related to oxidative stress. Acetaminophen intoxication and pulmonary fibrosis are models for use of NAC. Recent work on COPD appears to show a decrease in exacerbations, improvement in symptoms and quality-of-life, and perhaps a reduction in the alteration of ventilatory function.

Asunto(s)

Acetilcisteína/farmacología , Enfermedades Pulmonares/genética , Estrés Oxidativo , Glutatión/biosíntesis , Humanos , Inflamación , Enfermedades Pulmonares/inmunología

RESUMEN

Most human carcinoma cell lines lack the high-affinity receptors for adenovirus serotype 5 (Ad5) at their surface and are nonpermissive to Ad5. We therefore tested the efficiency of retargeting Ad5 to alternative cellular receptors via immunoglobulin (Ig)-binding domains inserted at the extremity of short-shafted, knobless fibers. The two recombinant Ad5's constructed, Ad5/R7-Z(wt)-Z(wt) and Ad5/R7-C2-C2, carried tandem Ig-binding domains from Staphylococcal protein A (abbreviated Z(wt)) and from Streptococcal protein G (C2), respectively. Both viruses bound their specific Ig isotypes with the expected affinity. They transduced human carcinoma cells independently of the CAR pathway, via cell surface receptors targeted by specific monoclonal antibodies, that is, EGF-R on A549, HT29 and SW1116, HER-2/neu on SK-OV-3 and SK-BR-3, CA242 (epitope recognized by the monoclonal antibody C242) antigen on HT29 and SW1116, and PSMA (prostate-specific membrane antigen) expressed on HEK-293 cells, respectively. However, Colo201 and Colo205 cells were neither transduced by targeting CA242 or EGF-R nor were LNCaP cells transduced by targeting PSMA. Our results suggested that one given surface receptor could mediate transduction of certain cells but not others, indicating that factors and steps other than cell surface expression and virus-receptor interaction are additional determinants of Ad5-mediated transduction of tumor cells. Using penton base RGD mutants, we found that one of these limiting steps was virus endocytosis.

Asunto(s)

Adenovirus Humanos/genética , Anticuerpos/metabolismo , Proteínas de la Cápside/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Neoplasias/terapia , Western Blotting/métodos , Línea Celular Transformada , Línea Celular Tumoral , Ingeniería Genética , Vectores Genéticos/genética , Humanos , Microscopía Electrónica , Unión Proteica , Transducción Genética/métodos , Virión/genética , Integración Viral

RESUMEN

The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

Asunto(s)

Adenoviridae/fisiología , Vectores Genéticos , Proteína Estafilocócica A/genética , Adenoviridae/química , Animales , Células COS , Técnicas de Transferencia de Gen , Humanos , Ligandos , Especificidad de Órganos , Pliegue de Proteína , Spodoptera , Proteína Estafilocócica A/química , Transducción Genética , Replicación Viral/genética

RESUMEN

Binding of bacteriophage T5 to Escherichia coli cells is mediated by specific interactions between the receptor-binding protein pb5 (67.8 kDa) and the outer membrane iron-transporter FhuA. A histidine-tagged form of pb5 was overproduced and purified. Isolated pb5 is monomeric and organized mostly as beta-sheets (51%). pb5 functionality was attested in vivo by its ability to impair infection of E. coli cells by phage T5 and Phi80, and to prevent growth of bacteria on iron-ferrichrome as unique iron source. pb5 was functional in vitro, since addition of an equimolar concentration of pb5 to purified FhuA prevented DNA release from phage T5. However, pb5 alone was not sufficient for the conversion of FhuA into an open channel. Direct interaction of pb5 with FhuA was demonstrated by isolating a pb5/FhuA complex using size-exclusion chromatography. The stoichiometry, 1 mol of pb5/1 mol of FhuA, was deduced from its molecular mass, established by analytical ultracentrifugation after determination of the amount of bound detergent. SDS-PAGE and differential scanning calorimetry experiments highlighted the great stability of the complex: (i) it was not dissociated by 2% SDS even when the temperature was raised to 70 degrees C; (ii) thermal denaturation of the complex occurred at 85 degrees C, while pb5 and FhuA were denatured at 45 degrees C and 74 degrees C, respectively. The stability of the complex renders it suitable for high-resolution structural studies, allowing future analysis of conformational changes into both FhuA and pb5 upon adsorption of the virus to its host.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Receptores Virales/química , Proteínas Virales/química , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Cromatografía en Gel , Dicroismo Circular , Estabilidad de Medicamentos , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/virología , Proteínas de Escherichia coli/genética , Canales Iónicos/química , Sustancias Macromoleculares , Desnaturalización Proteica , Receptores Virales/genética , Proteínas Recombinantes/química , Fagos T/química , Fagos T/genética , Temperatura , Termodinámica , Ultracentrifugación , Proteínas Virales/genética