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1.
Epilepsy Behav ; 115: 107685, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33360177

RESUMEN

The present study describes the development and preliminary psychometric evaluation of the Psychological Flexibility in Epilepsy Questionnaire (PFEQ), which was developed to measure the construct referred to as Psychological Flexibility (PF) in epilepsy. Results from 81 participants with epilepsy support a one-factor solution, consisting of 12 items, indicating a satisfactory structure and reliability with an alpha coefficient of 0.92. Criterion validity of the instrument was supported by moderate correlations with outcomes predicting quality of life, PF in the general population, depressive symptoms, and anxiety symptoms. Number of seizures in the past three and 12 months was positively correlated with scores on the PFEQ. These findings are consistent with the instrument's underlying theory. Further development and investigation of the PFEQ is recommended, as the preliminary results of the questionnaire suggest that the scale has the potential to be a valuable contribution to the future exploration of the role of PF in epilepsy.


Asunto(s)
Epilepsia , Calidad de Vida , Epilepsia/diagnóstico , Humanos , Psicometría , Reproducibilidad de los Resultados , Encuestas y Cuestionarios
2.
J Biotechnol ; 385: 23-29, 2024 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-38408644

RESUMEN

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.


Asunto(s)
ADN Cruciforme , Resolvasas de Unión Holliday , ADN Cruciforme/genética , Resolvasas de Unión Holliday/química , Resolvasas de Unión Holliday/genética , Resolvasas de Unión Holliday/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , ADN/genética , Oligonucleótidos , Digestión , Conformación de Ácido Nucleico
3.
Acta Crystallogr D Struct Biol ; 78(Pt 2): 212-227, 2022 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-35102887

RESUMEN

This study describes the production, characterization and structure determination of a novel Holliday junction-resolving enzyme. The enzyme, termed Hjc_15-6, is encoded in the genome of phage Tth15-6, which infects Thermus thermophilus. Hjc_15-6 was heterologously produced in Escherichia coli and high yields of soluble and biologically active recombinant enzyme were obtained in both complex and defined media. Amino-acid sequence and structure comparison suggested that the enzyme belongs to a group of enzymes classified as archaeal Holliday junction-resolving enzymes, which are typically divalent metal ion-binding dimers that are able to cleave X-shaped dsDNA-Holliday junctions (Hjs). The crystal structure of Hjc_15-6 was determined to 2.5 Šresolution using the selenomethionine single-wavelength anomalous dispersion method. To our knowledge, this is the first crystal structure of an Hj-resolving enzyme originating from a bacteriophage that can be classified as an archaeal type of Hj-resolving enzyme. As such, it represents a new fold for Hj-resolving enzymes from phages. Characterization of the structure of Hjc_15-6 suggests that it may form a dimer, or even a homodimer of dimers, and activity studies show endonuclease activity towards Hjs. Furthermore, based on sequence analysis it is proposed that Hjc_15-6 has a three-part catalytic motif corresponding to E-SD-EVK, and this motif may be common among other Hj-resolving enzymes originating from thermophilic bacteriophages.


Asunto(s)
Bacteriófagos , ADN Cruciforme , Archaea/genética , Archaea/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Resolvasas de Unión Holliday/química , Resolvasas de Unión Holliday/genética , Resolvasas de Unión Holliday/metabolismo , Thermus thermophilus
4.
Acta Crystallogr D Struct Biol ; 78(Pt 11): 1384-1398, 2022 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-36322421

RESUMEN

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SßαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SßαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SßαR motif, was first determined to 2.19 Šresolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Šresolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.


Asunto(s)
Bacteriófagos , ADN Polimerasa I , ADN Polimerasa I/química , ADN Polimerasa I/genética , Fosfodiesterasa I , Thermus , Polimerasa Taq/química , Escherichia coli
5.
FEMS Microbiol Lett ; 368(12)2021 06 24.
Artículo en Inglés | MEDLINE | ID: mdl-34114607

RESUMEN

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life.


Asunto(s)
Genoma Viral/genética , Metagenómica , Bioprospección/organización & administración , Biología Computacional , Bases de Datos Genéticas , Europa (Continente) , Respiraderos Hidrotermales/virología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo , Viroma/genética , Virus/clasificación , Virus/genética
6.
Cogn Behav Ther ; 37(3): 169-82, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18608312

RESUMEN

Although 14% to 42% of people with whiplash injuries end up with chronic debilitating pain, there is still a paucity of empirically supported treatments for this group of patients. In chronic pain management, there is increasing consensus regarding the importance of a behavioural medicine approach to symptoms and disability. Cognitive behaviour therapy has proven to be beneficial in the treatment of chronic pain. An approach that promotes acceptance of, or willingness to experience, pain and other associated negative private events (e.g. fear, anxiety, and fatigue) instead of reducing or controlling symptoms has received increasing attention. Although the empirical support for treatments emphasizing exposure and acceptance (such as acceptance and commitment therapy) is growing, there is clearly a need for more outcome studies, especially randomized controlled trials. In this study, participants (N = 21) with chronic pain and whiplash-associated disorders were recruited from a patient organization and randomized to either a treatment or a wait-list control condition. Both groups continued to receive treatment as usual. In the experimental condition, a learning theory framework was applied to the analysis and treatment. The intervention consisted of a 10-session protocol emphasizing values-based exposure and acceptance strategies to improve functioning and life satisfaction by increasing the participants' abilities to behave in accordance with values in the presence of interfering pain and distress (psychological flexibility). After treatment, significant differences in favor of the treatment group were seen in pain disability, life satisfaction, fear of movements, depression, and psychological inflexibility. No change for any of the groups was seen in pain intensity. Improvements in the treatment group were maintained at 7-month follow-up. The authors discuss implications of these findings and offer suggestions for further research in this area.


Asunto(s)
Actividades Cotidianas/psicología , Adaptación Psicológica , Terapia Cognitivo-Conductual , Desensibilización Psicológica , Dolor/psicología , Calidad de Vida/psicología , Lesiones por Latigazo Cervical/psicología , Accidentes de Tránsito/psicología , Adulto , Anciano , Enfermedad Crónica , Terapia Combinada , Depresión/psicología , Depresión/rehabilitación , Evaluación de la Discapacidad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Dolor/rehabilitación , Dimensión del Dolor , Rehabilitación Vocacional , Rol del Enfermo , Resultado del Tratamiento , Lesiones por Latigazo Cervical/rehabilitación
7.
J Biotechnol ; 122(2): 216-25, 2006 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-16442653

RESUMEN

A new chromatographic method based on affinity supermacroporous monolithic cryogels is developed for binding and analyzing inclusion bodies during fermentation. The work demonstrated that it is possible to bind specific IgG and IgY antibodies to the 15 and 17 amino acids at the terminus ends of a 33 kDa target protein aggregated as inclusion bodies. The antibody treated inclusion bodies from lysed fermentation broth can be specifically retained in protein A and pseudo-biospecific ligand sulfamethazine modified supermacroporous cryogels. The degree of binding of IgG and IgY treated inclusion bodies to the Protein A and sulfamethazine gels are investigated, as well as the influence of pH on the sulfamethazine ligand. Optimum binding of 78 and 72% was observed on both protein A and sulfamethazine modified cryogel columns, respectively, using IgG labeling of the inclusion bodies. The antibody treated inclusion bodies pass through unretained in the sulfamethazine supermacroporous gel at pH that does not favour the binding between the ligand on the gel and the antibodies on the surface of inclusion bodies. Also the unlabeled inclusion bodies went through the gel unretained, showing no non-specific binding or trapping within the gel. These findings may very well be the foundation for the building of a powerful analytical tool during fermentation of inclusion bodies as well as a convenient way to purify them from fermentation broth. These results also support our earlier findings [Kumar, A., Plieva, F.M., Galaev, I.Yu., Mattiasson, B., 2003. Affinity fractionation of lymphocytes using a monolithic cyogel. J. Immunol. Methods 283, 185-194] with mammalian cells that were surface labeled with specific antibodies and recognized on protein A supermacroporous gels. A general binding and separation system can be established on antibody binding cryogel affinity matrices.


Asunto(s)
Anticuerpos/química , Fraccionamiento Celular/métodos , Cromatografía de Afinidad/métodos , Cuerpos de Inclusión/química , Proteínas/aislamiento & purificación , Geles/química , Inmunoglobulina G/química , Inmunoglobulinas/química , Cuerpos de Inclusión/inmunología , Porosidad , Proteína Estafilocócica A/química , Sulfametazina/química
8.
Biosens Bioelectron ; 18(5-6): 547-53, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12706561

RESUMEN

A novel capacitance biosensor based on synthetic phytochelatins for sensitive detection of heavy metals is described. Synthetic phytochelatin (Glu-Cys)(20)Gly (EC20) fused to the maltose binding domain protein was expressed in Escherichia coli and purified for construction of the biosensor. The new biosensor was able to detect Hg(2+), Cd(2+), Pb(2+), Cu(2+) and Zn(2+) ions in concentration range of 100 fM-10 mM, and the order of sensitivity was S(Zn)>S(Cu)>S(Hg)>>S(Cd) congruent with S(Pb). The biological sensing element of the sensor could be regenerated using EDTA and the storage stability of the biosensor was 15 days.


Asunto(s)
Técnicas Biosensibles , Electroquímica/instrumentación , Análisis de Falla de Equipo , Metaloproteínas/biosíntesis , Metaloproteínas/química , Metales Pesados/análisis , Metales Pesados/química , Capacidad Eléctrica , Electroquímica/métodos , Diseño de Equipo , Escherichia coli/genética , Escherichia coli/metabolismo , Glutatión , Metaloproteínas/genética , Microquímica/instrumentación , Microquímica/métodos , Fitoquelatinas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
9.
Anal Biochem ; 354(2): 229-37, 2006 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-16729960

RESUMEN

A novel minicolumn chromatographic method to monitor the production of inclusion bodies during fermentation and an enzyme-linked immunosorbent assay (ELISA) system allowing direct analysis of the particles with surface-displayed antigens are described. A 33-kDa protein containing 306 amino acids with three sulfur bridges produced as inclusion bodies was labeled with polyclonal antibodies against 15 amino acid (anti-A15) and 17 amino acid (anti-B17) residues at the N- and C-terminal ends of the protein, respectively. Labeled particles were bound to macroporous monolithic protein A-cryogel adsorbents inserted into the open-ended wells of a 96-well plate (referred to as protein A-cryogel minicolumn plate). The concept behind this application is that the binding degree of inclusion bodies from lysed fermentation broth to the cryogel minicolumns increases with an increase in their concentration during fermentation. The technique allowed us to monitor the increase in the production levels of the inclusion bodies as the fermentation process progressed. The system also has a built-in quality parameter to ensure that the target protein has been fully expressed. Alternatively, inclusion bodies immobilized on phenyl-cryogel minicolumn plate were used in indirect ELISA based on anti-A15 and anti-B17 antibodies against terminal amino acid residues displayed on the surface of inclusion bodies. Drainage-protected properties of the cryogel minicolumns allow performance of successive reactions with tested immunoglobulin G (IgG) samples and enzyme-conjugated secondary IgG and of enzymatic reaction within the adsorbent.


Asunto(s)
Cromatografía/métodos , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Adsorción , Anticuerpos Antibacterianos , Reactores Biológicos , Proteínas Sanguíneas , Cromatografía/instrumentación , Criogeles , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/inmunología , Fermentación , Fibronectinas , Citometría de Flujo , Hidrogeles , Microscopía Electrónica de Rastreo , Proteína Estafilocócica A
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