Unexpected exosites on the surface of matrix metalloproteinase-12 that fine-tune specificity for elastin and collagen V

Abstract only availableMMP-12 hydrolyzes collagens and elastin in normal physiological processes of development, wound repair, and reconstruction of the extracellular matrix. However, chronically elevated levels of MMP-12 activity can sustain disease states in certain tissues. Excessive MMP-12 activity upon elastic fibrils of lungs and arteries causes inflammation at those sites, resulting in emphysema, while excessive activity upon collagens triggers rupture of atheromatous plaques, which can lead to heart attack or stroke. Thus, research into the binding site interactions between MMP-12 and macromolecular substrates provides surface characterization of the enzyme and expedites the process of designing artificial inhibitor molecules to selectively inactivate MMP-12. The catalytic domain of MMP-12 spans amino acid residue 100 to 263, and this region is particularly important during hydrolysis of substrates. Three secondary binding sites within the catalytic domain were examined: phenylalanine 202 (F202), threonine 205 (T205), and histidine 206 (H206). Site-directed mutagenesis was performed at the aforementioned exosites to obtain single point mutations glutamine 202 (F202Q), lysine 205 (T205K), and histidine 206 (H206D). The MMP-12 mutants were expressed as the 18.2kDa catalytic domain in BL21 derived E. coli Rosetta™ 2 host strains. The mutated MMP-12 enzymes were isolated as insoluble inclusion bodies after lysis of E. coli cells using the French press. The inclusion bodies were solubilized in neutral 6M urea buffer, and preliminary purification with cation-exchange chromatography yielded a nucleic acid-free fraction of denatured enzyme. Refolding of the MMP-12 mutants was done by dialysis with serially diluted urea solution containing Tris-HCl buffer and CaCl2 at pH 7.5 without Zn++. ZnCl2 was added to the final dialysis buffer to complete the refolding process. Final purification of active renatured enzyme was achieved by another cation exchange chromatography run. Then, a Bio-Rad protein assay determined the concentration of the three MMP-12 mutants. Two types of flurometric-based kinetic experiments were performed to study changes in catalytic activity of the three MMP-12 mutants versus wild-type MMP-12: active site titration and substrate activity. Active site titration quantified the concentration of active sites for each MMP-12 mutant. The substrate runs for the substrates FS-6, fluorescently labeled elastin, and a triple helical peptide mimic of collagen V (THP-V) produced raw kinetics data, in the form of progress curves. Microcal Origin Pro 7.5 was used to analyze the data to give the kcat, the number of times each enzyme site converts substrate to product per second, and the Km, the concentration needed to achieve one half Vmax. Furthermore, dividing Kcat by Km revealed the amount of activity upon FS-6, elastin, and THP-V for each MMP-12 mutant with FS-6 as the control for mutated and wild-type MMP-12. Since the kcat/Km for wild-type MMP-12 was already known, comparison of activity upon THP-V and elastin between wild type MMP-12 and each MMP-12 mutant was achieved. Ongoing data collection suggests diminished catalytic activity upon elastin and THP-V in all three MMP-12 mutants (F202Q, T205K, and H206D) when compared to wild-type MMP-12, while showing no decrease in activity upon the general MMP substrate, FS-6. To date, data suggests that all three exosites are individually involved in fine-tuning MMP-12 specificity for elastin and triple helical peptide mimics of collagen V.NSF-REU Program in Biological Sciences & Biochemistr

Emotion regulation strategies in bipolar disorder: A systematic and critical review

Background Theoretical frameworks emphasise associations between interpretations and responses to affect and bipolar disorder (BD). This review (PROSPERO CRD42016043801) investigated which emotion regulation (ER) strategies have been applied to BD, are elevated in BD compared to clinical and non-clinical controls, and are associated with clinical and functional outcomes in BD Methods Search terms relating to emotion regulation, coping and bipolar disorder were entered into Embase, MedLine and PsycInfo. Quantitative studies investigating relationships between ER strategies and BD were eligible for this narrative synthesis Results A large volume of research (n = 47) investigated specific ER strategies in BD. Maladaptive strategies such as rumination and dampening were elevated in BD compared to controls and these particular strategies had a detrimental impact on outcomes such as mood symptoms. BD had a similar profile of ER strategies to unipolar depression, but there was limited comparison to other clinical groups. People with BD did not generally have deficits in using adaptive strategies, as evidenced by comparisons with controls and experimental studies Limitations Methodological heterogeneity and a lack of ecologically valid ER assessments Conclusions Empirical literature is critiqued in line with contemporary theories of BD and of emotion regulation more generally, in order to inform future research recommendations. This includes investigation of the importance of context in the impact of ER strategies, and discrepancies between trait and state use of ER strategies, particularly through experience sampling

Elastography of multicellular aggregates submitted to osmo-mechanical stress

International audienceTumors are subjected to mechanical stress generated by their own growth in a confined environment, and by their surrounding tissues. Recent works have focused on the study of the growth of spherical aggregates of cells, spheroids, under controlled confinement or stress. In this study we demonstrate the measurement of spatially and temporally resolved deformation maps inside spheroids while applying an osmo-mechanical stress. We use full field optical coherence tomography, a high resolution imaging technique well suited for real-time measurements of deformation in living tissues under stress. Using the spherical symmetry of the experiment, we compare our data to a mechanical modeling of the spheroid as a continuous medium. We estimate the viscoelastic parameters of spheroids and discuss the apparent tissue anisotropy after the osmo-mechanical stress

Saúde bucal: aspectos básicos e atenção ao adulto

Este módulo integra o curso de Especialização em Atenção Básica em Saúde da Família. Tem como objetivo oferecer para os profissionais em formação, subsídios teóricos para a compreensão da saúde bucal do adulto, instrumentalizando-o para a tomada de decisão frente aos problemas do município. Tem como referência para a organização dos serviços, os indivíduos acima de 18 anos - o adulto jovem - até as pessoas com 59 anos de idade. Ao final do estudo deste módulo, espera-se que o aluno seja capaz de conhecer as políticas públicas na área da saúde bucal; identificar sob a ótica epidemiológica os principais agravos da cavidade bucal em adultos; identificar as causas dos principais agravos da saúde bucal do adulto; estabelecer uma estratégia adequada para o enfrentamento dos problemas de saúde bucal na área de atuação e; definir as ações que poderão trazer resultados satisfatórios para a saúde bucal da população adulta.Banco Nacional de Desenvolvimento Econômico e Social - BNDESMinistério da Saúd

Technical aspects of an impact acceleration traumatic brain injury rat model with potential suitability for both microdialysis and PtiO2 monitoring

This report describes technical adaptations of a traumatic brain injury (TBI) model-largely inspired by Marmarou-in order to monitor microdialysis data and PtiO2 (brain tissue oxygen) before, during and after injury. We particularly focalize on our model requirements which allows us to re-create some drastic pathological characteristics experienced by severely head-injured patients: impact on a closed skull, no ventilation immediately after impact, presence of diffuse axonal injuries and secondary brain insults from systemic origin...We notably give priority to minimize anaesthesia duration in order to tend to banish any neuroprotection. Our new model will henceforth allow a better understanding of neurochemical and biochemical alterations resulting from traumatic brain injury, using microdialysis and PtiO2 techniques already monitored in our Intensive Care Unit. Studies on efficiency and therapeutic window of neuroprotective pharmacological molecules are now conceivable to ameliorate severe head-injury treatment

Zener transitions between dissipative Bloch bands. II: Current Response at Finite Temperature

We extend, to include the effects of finite temperature, our earlier study of the interband dynamics of electrons with Markoffian dephasing under the influence of uniform static electric fields. We use a simple two-band tight-binding model and study the electric current response as a function of field strength and the model parameters. In addition to the Esaki-Tsu peak, near where the Bloch frequency equals the damping rate, we find current peaks near the Zener resonances, at equally spaced values of the inverse electric field. These become more prominenent and numerous with increasing bandwidth (in units of the temperature, with other parameters fixed). As expected, they broaden with increasing damping (dephasing).Comment: 5 pages, LateX, plus 5 postscript figure

Corruption and Development

SUMMARY Corruption does not necessarily either hinder or encourage development and even when it helps, it is not thereby legitimised. Development, however, in the form now favoured of State control of the economy, does encourage corruption. This should be accepted as a necessary cost, with vigorous efforts made to reduce it. RESUME Corruption et développement On ne peut pas dire que la corruption entrave ou encourage forcément le développement, mais même si elle l'aide, elle n'est pas pour cela légitime. Par contre, le développement, tel qu'on le préfère aujourd'hui sous la forme de contrôle de l'économie par l'état, encourage la corruption. Tout en faisant tout pour la réduire, il faut l'accepter comme un mal nécessaire. RESUMEN Corrupción y Desarrollo No siempre sucede que la corrupción impida o anime el desarrollo, e incluso en aquellos casos en que es beneficioso, no llega a ser legitimo. Sin embargo, la corrupción se ve alentada en las condiciones de desarrollo actualmente favorecidas mediante un control estatal en la economía. Se debe aceptar esto como un costo necesario, haciendo todo lo posible para reducirlo

SIN PALABRAS: La comunicación NO verbal en estudiantes de profesiones sanitarias y no sanitarias

La Comunicación NO Verbal (CNV) es un tipo de comunicación que no siempre se incluye como tal en las competencias y objetivos de las titulaciones académicas y que a menudo se considera sólo desde un punto de vista teórico. Pero, en el ámbito laboral, se exige a los profesionales que sepan controlar a su favor la CNV, en especial a los profesionales de la salud, por el posible impacto sobre el paciente. Mediante formulario on-line, se pidió a los estudiantes matriculados en titulaciones relacionadas y no relacionadas con la sanidad, que valorarán el papel de la CNV en diferentes situaciones de la vida cotidiana. Participaron estudiantes universitarios: U. de Alicante y U. Nacional de Tucumán (Argentina), y de Formación Profesional: IES Leonardo da Vinci (Alicante) e INS Gabriela Mistral (Sant Vicenç dels Horts, Barcelona). Los resultados muestran que los estudiantes universitarios consideran más importante y valoran mejor la CNV que los de Formación Profesional. Sin embargo, no se aprecian diferencias significativas entre estudiantes de titulaciones sanitarias y no sanitarias en ninguno de los dos niveles académicos. Ante estos resultados hacemos una llamada a la reflexión sobre la importancia que debería adquirir la práctica de la CNV en las titulaciones sanitarias