Microfluidic platforms for the characterization of in meso membrane protein crystallization

Membrane proteins, which reside in the membranes of cells, play a critical role in many important biological processes including cellular signaling, immune response, and material and energy transduction. Because of their key role in maintaining the environment within cells and facilitating intercellular interactions, understanding the function of these proteins is of tremendous medical and biochemical significance. Indeed, the malfunction of membrane proteins has been linked to numerous diseases including diabetes, cirrhosis of the liver, cystic fibrosis, cancer, Alzheimer's disease, hypertension, epilepsy, cataracts, tubulopathy, leukodystrophy, Leigh syndrome, anemia, sensorineural deafness, and hypertrophic cardiomyopathy.1-3 However, the structure of many of these proteins and the changes in their structure that lead to disease-related malfunctions are not well understood. Additionally, at least 60% of the pharmaceuticals currently available are thought to target membrane proteins, despite the fact that their exact mode of operation is not known.4-6 Developing a detailed understanding of the function of a protein is achieved by coupling biochemical experiments with knowledge of the structure of the protein. Currently the most common method for obtaining three-dimensional structure information is X-ray crystallography. However, no a priori methods are currently available to predict crystallization conditions for a given protein.7-14 This limitation is currently overcome by screening a large number of possible combinations of precipitants, buffer, salt, and pH conditions to identify conditions that are conducive to crystal nucleation and growth.7,9,11,15-24 Unfortunately, these screening efforts are often limited by difficulties associated with quantity and purity of available protein samples. While the two most significant bottlenecks for protein structure determination in general are the (i) obtaining sufficient quantities of high quality protein samples and (ii) growing high quality protein crystals that are suitable for X-ray structure determination,7,20,21,23,25-47 membrane proteins present additional challenges. For crystallization it is necessary to extract the membrane proteins from the cellular membrane. However, this process often leads to denaturation. In fact, membrane proteins have proven to be so difficult to crystallize that of the more than 66,000 structures deposited in the Protein Data Bank,48 less than 1% are for membrane proteins, with even fewer present at high resolution (< 2Å)4,6,49 and only a handful are human membrane proteins.49 A variety of strategies including detergent solubilization50-53 and the use of artificial membrane-like environments have been developed to circumvent this challenge.43,53-55 In recent years, the use of a lipidic mesophase as a medium for crystallizing membrane proteins has been demonstrated to increase success for a wide range of membrane proteins, including human receptor proteins.54,56-62 This in meso method for membrane protein crystallization, however, is still by no means routine due to challenges related to sample preparation at sub-microliter volumes and to crystal harvesting and X-ray data collection. This dissertation presents various aspects of the development of a microfluidic platform to enable high throughput in meso membrane protein crystallization at a level beyond the capabilities of current technologies. Microfluidic platforms for protein crystallization and other lab-on-a-chip applications have been well demonstrated.9,63-66 These integrated chips provide fine control over transport phenomena and the ability to perform high throughput analyses via highly integrated fluid networks. However, the development of microfluidic platforms for in meso protein crystallization required the development of strategies to cope with extremely viscous and non-Newtonian fluids. A theoretical treatment of highly viscous fluids in microfluidic devices is presented in Chapter 3, followed by the application of these strategies for the development of a microfluidic mixer capable of preparing a mesophase sample for in meso crystallization at a scale of less than 20 nL in Chapter 4. This approach was validated with the successful on chip in meso crystallization of the membrane protein bacteriorhodopsin. In summary, this is the first report of a microfluidic platform capable of performing in meso crystallization on-chip, representing a 1000x reduction in the scale at which mesophase trials can be prepared. Once protein crystals have formed, they are typically harvested from the droplet they were grown in and mounted for crystallographic analysis. Despite the high throughput automation present in nearly all other aspects of protein structure determination, the harvesting and mounting of crystals is still largely a manual process. Furthermore, during mounting the fragile protein crystals can potentially be damaged, both from physical and environmental shock. To circumvent these challenges an X-ray transparent microfluidic device architecture was developed to couple the benefits of scale, integration, and precise fluid control with the ability to perform in situ X-ray analysis (Chapter 5). This approach was validated successfully by crystallization and subsequent on-chip analysis of the soluble proteins lysozyme, thaumatin, and ribonuclease A and will be extended to microfluidic platforms for in meso membrane protein crystallization. The ability to perform in situ X-ray analysis was shown to provide extremely high quality diffraction data, in part as a result of not being affected by damage due to physical handling of the crystals. As part of the work described in this thesis, a variety of data collection strategies for in situ data analysis were also tested, including merging of small slices of data from a large number of crystals grown on a single chip, to allow for diffraction analysis at biologically relevant temperatures. While such strategies have been applied previously,57,59,61,67 they are potentially challenging when applied via traditional methods due to the need to grow and then mount a large number of crystals with minimal crystal-to-crystal variability. The integrated nature of microfluidic platforms easily enables the generation of a large number of reproducible crystallization trials. This, coupled with in situ analysis capabilities has the potential of being able to acquire high resolution structural data of proteins at biologically relevant conditions for which only small crystals, or crystals which are adversely affected by standard cryocooling techniques, could be obtained (Chapters 5 and 6). While the main focus of protein crystallography is to obtain three-dimensional protein structures, the results of typical experiments provide only a static picture of the protein. The use of polychromatic or Laue X-ray diffraction methods enables the collection of time resolved structural information. These experiments are very sensitive to crystal quality, however, and often suffer from severe radiation damage due to the intense polychromatic X-ray beams. Here, as before, the ability to perform in situ X-ray analysis on many small protein crystals within a microfluidic crystallization platform has the potential to overcome these challenges. An automated method for collecting a "single-shot" of data from a large number of crystals was developed in collaboration with the BioCARS team at the Advanced Photon Source at Argonne National Laboratory (Chapter 6). The work described in this thesis shows that, even more so than for traditional structure determination efforts, the ability to grow and analyze a large number of high quality crystals is critical to enable time resolved structural studies of novel proteins. In addition to enabling X-ray crystallography experiments, the development of X-ray transparent microfluidic platforms also has tremendous potential to answer other scientific questions, such as unraveling the mechanism of in meso crystallization. For instance, the lipidic mesophases utilized during in meso membrane protein crystallization can be characterized by small angle X-ray diffraction analysis. Coupling in situ analysis with microfluidic platforms capable of preparing these difficult mesophase samples at very small volumes has tremendous potential to enable the high throughput analysis of these systems on a scale that is not reasonably achievable using conventional sample preparation strategies (Chapter 7). In collaboration with the LS-CAT team at the Advanced Photon Source, an experimental station for small angle X-ray analysis coupled with the high quality visualization capabilities needed to target specific microfluidic samples on a highly integrated chip is under development. Characterizing the phase behavior of these mesophase systems and the effects of various additives present in crystallization trials is key for developing an understanding of how in meso crystallization occurs. A long term goal of these studies is to enable the rational design of in meso crystallization experiments so as to avoid or limit the need for high throughput screening efforts. In summary, this thesis describes the development of microfluidic platforms for protein crystallization with in situ analysis capabilities. Coupling the ability to perform in situ analysis with the small scale, fine control, and the high throughput nature of microfluidic platforms has tremendous potential to enable a new generation of crystallographic studies and facilitate the structure determination of important biological targets. The development of platforms for in meso membrane protein crystallization is particularly significant because they enable the preparation of highly viscous mixtures at a previously unachievable scale. Work in these areas is ongoing and has tremendous potential to improve not only current the methods of protein crystallization and crystallography, but also to enhance our knowledge of the structure and function of proteins which could have a significant scientific and medical impact on society as a whole. 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Kim jestem? Kim się staję? Od praktyki do teorii

W artykule w zakresie metody teologiczno-fundamentalnej przedstawione zostały następujące główne zagadnienia. 1. Konspekt edukacyjno-psychologicznych warsztatów, 2. Uzasadnienie metodyczne i metodologiczne przyjętej konstrukcji. W konspekcie szczegółowo opisane zostały kolejne kroki, które powinien podjąć prowadzący z pewnymi sugestiami ku czemu dane działania prowadzą. Szerzej zostały podane wnioski, do których prowadzący powinien podprowadzić uczestników warsztatów w obszarze teologii fundamentalnej – jako pomoc w poszukiwaniach celu, całości i sensu – wykorzystano elementy psychologii, filozofii, teologii duchowości. Procedura ćwiczeń przeprowadza uczestnika od pytań o samego siebie do pytań o siebie poprzez relacje z drugim, tak by uświadomić mu możliwość kontaktu z Drugim. W dalszej części przedstawienia opisano próbę odpowiedzi na pytanie „Co było potrzebne, od strony metody, by ten konspekt mógł powstać?” Koniecznym było skonstruowanie swoistej metafory i opracowano narrację, które razem stały się hipotezą wyjaśniającą obejmującą szerokie spektrum badanych relacji, pomiędzy konspektem i procesem jego powstawania a jego twórcą, procesem przeprowadzonych warsztatów a czytelnikiem, prowadzącym warsztaty a uczestnikiem. Na zakończenie przedstawiono ewaluację przeprowadzonych warsztatów.

Kim jestem? Kim się staję? W dialogicznym napięciu JA-TY. Źródła teoretyczne

W niniejszym artykule przedstawiono komponenty wchodzące w skład źródeł teoretycznych, będących podstawą powstania konspektu warsztatów. Treść artykułu nie wychodzi poza obszar metodologii teologicznej. Badania ukazujące przesłanki procesów zaaranżowanych w czasie warsztatów oraz podane wnioski końcowe wynikają z badań przeprowadzonych w polu analiz teologii fundamentalnej. Ta dziedzina teologiczna zakłada interdyscyplinarne łączenie metod i osiągnięć teologii i nauk antropologicznych. W kolejnych odsłonach ukazane zostały trzy aspekty odkrywania ludzkiego JA w dialogicznym napięciu z TY. Dzięki analizie transakcyjnej Erica Berne’a można ukazać, przy pomocy prostych technik edukacyjnych, że człowiek w poznaniu siebie potrzebuje drugiego w każdym aspekcie swego istnienia. Tym, co umożliwia mu bycie z sobą i innymi do zdolność wczucia. Tę kategorię badawczą analizowała Edyta Stein jako podstawę realnych relacji JA-TY, gdzie poprzez TY może zostać określony sam człowiek jako nie-ja, inny, drugi, czy ostatecznie osobowy Bóg. Możliwość zastosowania procesów opartych na wczuciu w przestrzeni relacji wiary doprowadza ku teologii duchowości. W jej ramach wypracowany został model antropologiczny, który widzi człowieka w ciągłym stawaniu się wobec innych i Boga. Przyjęta do badań praktyczno-biblijna antropologia Ignacego Loyoli pozwala na uwiarygodnienie kroków podjętych w czasie warsztatów, tak by nie były manipulacją a służyły wolnemu wyborowi uczestników w świadomym poszukiwaniu sensu i celu istnienia.

In vitro antioxidative activity of moss extract, and effect of moss on serum lipid level of mice fed with high-fat diet

Purpose: To evaluate the potential of active compounds derived from moss in the prevention and treatment of various diseases. Methods: Three species of moss were extracted with deionized water at 95 °C, and with 70.5 % ethanol at 85 °C. Analysis of total phenolic contents (TPC) of the extracts were performed by FolinCiocalteu (FC) method. The antioxidant activity of the extracts were determined using three methods, namely, by 2,2\'-azino-bis(3-ethylbenzothiazoline-6-sulphonic) acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). In vivo effects were evaluated in mice fed high fat diet (HFD) supplemented with 20 % ground moss. Cholesterol levels in HFD were evaluated by ophthalaldehyde method. Serum triglyceride levels were measured using triglyceride (TG) kit, while blood insulin level and leptin concentration were measured by enzyme-linked immunosorbent assay (ELISA) kit. Results: The moss extracts exhibited antioxidative effects, as evidenced of . TPC of 47.20 ± 11.20 to 119.87 ± 11.51 mg GAE/mg, respectively. ABTS scavenging activity was 1078.11 ± 18.95 to 2587.33 ± 46.19 μmol Trolox/mg, DPPH scavenging activity of were 42.11 ± 8.22 to 298.78 ± 20.02 μmol Trolox/mg, and FRAP value of 393.19 ± 24.64 to 1070.14 ± 17.92 μmol Trolox/mg, respectively. Mice fed with 20 % ground moss did not show any significant effect (p < 0.05) on visceral weight and blood lipid levels of HFD, while leptin concentrations reduced significantly to 4.74 ± 0.00 and 0.20 ± 0.00 ng/dL) relative to HFD alone (26.72 ± 6.53 ng/dL). Conclusion: Moss can potentially be used as an antioxidative ingredient, for the improvement of overall human health, suggesting that important medical benefits associated with moss consumption. However, further investigations are required to ascertain this.

La notion de l’explosion de la coopération et ses implications chez Kim Sterelny.

Le concept de coopération est souvent utilisé dans le domaine de l’éthique et de la politique pour illustrer et comprendre l’alignement des comportements associatifs entre les êtres humains. En lien avec ce concept, notre recherche portera sur la première question de savoir si Kim Sterelny (2003) réussit à produire un modèle théorique permettant d’expliquer les origines et les mécanismes de la coopération humaine. Notre recherche portera aussi sur la deuxième question de savoir s’il arrive à se servir de ce modèle pour infirmer la thèse de la modularité massive. Ainsi, ce mémoire traitera successivement du problème de la coopération, de la théorie de la sélection de groupe, du déclencheur écologique de la coopération des hominidés, des notions de coalition, d’exécution et d’engagement et finalement de la thèse de la modularité massive. Par l’examen de ces sujets, nous souhaitons démontrer que Sterelny n’arrive qu’à fournir une esquisse probable des origines et du développement de la coopération humaine et que sa critique de la thèse de la modularité massive n’arrive pas à infirmer cette dernière.

La notion de l’explosion de la coopération et ses implications chez Kim Sterelny

Le concept de coopération est souvent utilisé dans le domaine de l’éthique et de la politique pour illustrer et comprendre l’alignement des comportements associatifs entre les êtres humains. En lien avec ce concept, notre recherche portera sur la première question de savoir si Kim Sterelny (2003) réussit à produire un modèle théorique permettant d’expliquer les origines et les mécanismes de la coopération humaine. Notre recherche portera aussi sur la deuxième question de savoir s’il arrive à se servir de ce modèle pour infirmer la thèse de la modularité massive. Ainsi, ce mémoire traitera successivement du problème de la coopération, de la théorie de la sélection de groupe, du déclencheur écologique de la coopération des hominidés, des notions de coalition, d’exécution et d’engagement et finalement de la thèse de la modularité massive. Par l’examen de ces sujets, nous souhaitons démontrer que Sterelny n’arrive qu’à fournir une esquisse probable des origines et du développement de la coopération humaine et que sa critique de la thèse de la modularité massive n’arrive pas à infirmer cette dernière.

Prevalencia de síntomas de fatiga crónica / encefalomielitis miálgica (SFC/EM) y su relación con factores ocupacionales en trabajadores en una empresa de vigilancia en Bogotá, Colombia, 2016

Antecedentes: El síndrome de fatiga crónica/encefalomielitis miálgica (SFC/EM), un trastorno debilitante y complejo que se caracteriza por un cansancio intenso, ha sido estudiado en población general, sin embargo, su exploración en población trabajadora ha sido limitada. Objetivo: Determinar la prevalencia de síntomas asociados a SFC/EM y su relación con factores ocupacionales en personal de una empresa de vigilancia en Bogotá, durante el año 2016. Materiales y métodos: Estudio de corte transversal en una empresa de vigilancia, utilizando como instrumento para la recolección de datos la historia clínica-ocupacional. En las variables cualitativas se obtuvieron frecuencias simples y porcentajes y en las variables cuantitativas medidas de tendencia central y de dispersión. Se determinaron asociaciones entre variables (Ji-cuadrado de Pearson o test exacto de Fisher, valores esperados <5), (mann-whitney.y un modelo de regresión logística incondicional (p<0.05)). Resultados: Se evaluaron 162 trabajadores, los síntomas de SFC/EM con mayor prevalencia fueron sueño no reparador (38,3%) y dolor muscular (30,2%). Se encontró asociación estadísticamente significativa entre fatiga severa y crónica por al menos 6 meses con alteración en sistema nervioso (p=0,016) y consumo de medicamentos (p=0,043), así mismo entre el sueño no reparador con el número de horas de sueño de 5 a 7 horas (p=0,002). Conclusión: En los vigilantes el síntoma de SFC/EM más prevalente fue sueño no reparador y este se asoció con el número de horas de sueño de 5 a 7 horas. Con el estudio se pudieron determinar los casos probables de SFC/EM los cuales se beneficiarían de una valoración médica integral para un diagnóstico oportuno.

Impacto de los Niveles de Tacrolimus en el Rechazo al Injerto y la Tasa de Filtración Glomerular en Pacientes con Trasplante Hepático realizado en la FCI-IC entre 2009 y 2014

Introducción: El tacrolimus es el medicamento de elección para evitar el rechazo al injerto hepático. Su dosis se ajusta a partir de los niveles séricos que se toman periódicamente para asegurar rango terapéutico. Además, niveles elevados se asocian con disfunción renal postrasplante. Sin embargo, no hay consenso frente a los niveles adecuados para pacientes con trasplante hepático. Objetivo: Determinar la relación entre los niveles de tacrolimus y la presencia de rechazo agudo al injerto hepático en pacientes con trasplante hepático realizado en la Fundación Cardioinfantil – Instituto de Cardiología (FCI-IC). Determinar la relación entre los niveles de tacrolimus y la TFG en pacientes con trasplante hepático realizado en la FCI-IC. Métodos: Estudio observacional tipo cohorte histórica en pacientes adultos con trasplante hepático realizado en la FCI-IC entre 2009-2014. Resultados: No se encontró una asociación estadísticamente significativa entre los niveles de tacrolimus y la presencia de rechazo agudo, en sus diferentes definiciones (OR=1,02, p=0,14 y OR=1,01, p=0,29) incluso al ajustar por otras covariables (OR=1,03, p=0,10 y OR=1,02, p=0,25). No fue posible corroborar el diagnóstico con biopsia porque no todos la tenían. Si bien la relación entre los niveles de tacrolimus y la TFG fue estadísticamente significativa (p≤0,001), tiene bajo impacto clínico, pues la TFG disminuyó menos de un punto por cada incremento en 1 ng/ml en los niveles de tacrolimus. Conclusiones: Se necesitan más estudios para establecer la relación entre la exposición a tacrolimus y estos desenlaces para definir si es seguro disminuir su dosis con el fin de reducir los eventos adversos.

FAST-E en Pacientes con Trauma Abdominal Cerrado Estable, en un Departamento de Urgencias en Colombia

El impacto que ha generado el trauma en Colombia a lo largo de la historia, nos ha obligado a mejorar y adaptar diferentes tipos de sistemas de atención en trauma, basados en los lineamientos internacionales, los cuales buscan evitar el significativo aumento en las tasas de mortalidad y discapacidad que se obtienen de este, especialmente en los servicios de Emergencias en los cuales se reciben el 100% de estos pacientes con traumatismo múltiple o politraumatismo. Dentro de este grupo de pacientes hay un subgrupo que son las pacientes con trauma de abdomen que cursan con estabilidad hemodinámica y además son clasificados de bajo riesgo, ya sea por índices de trauma o por otros métodos como la medición sérica de lactato, los cuales tienen un papel poco despreciable al momento de ver mortalidad y discapacidad por trauma, ya sea penetrante o cerrado; en este trabajo específicamente nos centramos en las personas que consultan al servicio de Emergencias con trauma cerrado de abdomen los cuales son considerados de bajo riesgo, siendo este subgrupo de pacientes uno de los más difíciles de abordar y enfocar al momento de la valoración inicial, ya que se debe tener la seguridad de que no hay lesiones que comprometen la vida y por consiguiente estos pacientes puedan ser dados de alta.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Cardiac Magnetic Resonance Assessment Of Interstitial Myocardial Fibrosis And Cardiomyocyte Hypertrophy In Hypertensive Mice Treated With Spironolactone.

Nearly 50% of patients with heart failure (HF) have preserved LV ejection fraction, with interstitial fibrosis and cardiomyocyte hypertrophy as early manifestations of pressure overload. However, methods to assess both tissue characteristics dynamically and noninvasively with therapy are lacking. We measured the effects of mineralocorticoid receptor blockade on tissue phenotypes in LV pressure overload using cardiac magnetic resonance (CMR). Mice were randomized to l-nitro-ω-methyl ester (l-NAME, 3 mg/mL in water; n=22), or l-NAME with spironolactone (50 mg/kg/day in subcutaneous pellets; n=21). Myocardial extracellular volume (ECV; marker of diffuse interstitial fibrosis) and the intracellular lifetime of water (τic; marker of cardiomyocyte hypertrophy) were determined by CMR T1 imaging at baseline and after 7 weeks of therapy alongside histological assessments. Administration of l-NAME induced hypertensive heart disease in mice, with increases in mean arterial pressure, LV mass, ECV, and τic compared with placebo-treated controls, while LV ejection fraction was preserved (>50%). In comparison, animals receiving both spironolactone and l-NAME (l-NAME+S) showed less concentric remodeling, and a lower myocardial ECV and τic, indicating decreased interstitial fibrosis and cardiomyocyte hypertrophy (ECV: 0.43 ± 0.09 for l-NAME versus 0.25 ± 0.03 for l-NAME+S, P<0.001; τic: 0.42 ± 0.11 for l-NAME groups versus 0.12 ± 0.05 for l-NAME+S group). Mice treated with a combination of l-NAME and spironolactone were similar to placebo-treated controls at 7 weeks. Spironolactone attenuates interstitial fibrosis and cardiomyocyte hypertrophy in hypertensive heart disease. CMR can phenotype myocardial tissue remodeling in pressure-overload, furthering our understanding of HF progression.

15-deoxy-Δ12,14-prostaglandin J2 Reduces Albumin-induced Arthritis In Temporomandibular Joint Of Rats.

The aim of this study was to evaluate the peripheral effect of 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) in albumin-induced arthritis in temporomandibular joint (TMJ) of rats. Antigen-induced arthritis (AIA) was generated in rats with methylated bovine serum albumin (mBSA) diluted in complete Freund׳s adjuvant. Pretreatment with an intra-articular injection of 15d-PGJ2 (100 ng/TMJ) before mBSA intra-articular injection (10 µg/TMJ) (challenge) in immunized rats significantly reduced the albumin-induced arthritis inflammation. The results demonstrated that 15d-PGJ2 was able to inhibit plasma extravasation, leukocyte migration and the release of inflammatory cytokines IL-6, IL-12, IL-18 and the chemokine CINC-1 in the TMJ tissues. In addition, 15d-PGJ2 was able to increase the expression of the anti-adhesive molecule CD55 and the anti-inflammatory cytokine IL-10. Taken together, it is possible to suggest that 15d-PGJ2 inhibit leukocyte infiltration and subsequently inflammatory process, through a shift in the balance of the pro- and anti-adhesive properties. Thus, 15d-PGJ2 might be used as a potential anti-inflammatory drug to treat arthritis-induced inflammation of the temporomandibular joint.

Correlation Between Testosterone And Psa Kinetics In Metastatic Prostate Cancer Patients Treated With Diverse Chemical Castrations.

To assess total testosterone and prostatic-specific antigen (PSA) kinetics among diverse chemical castrations, advanced-stage prostate cancer patients were randomized into three groups of 20: Group 1, Leuprolide 3.75 mg; Group 2, Leuprolide 7.5 mg; and Group 3, Goserelin 3.6 mg. All groups were treated with monthly application of the respective drugs. The patients' levels of serum total testosterone and PSA were evaluated at two time periods: before the treatment and 3 months after the treatment. Spearman's rank correlation coefficient was utilized to verify the hypothesis of linear correlation between total testosterone and PSA levels. At the beginning the patients' age, stage, grade, PSA, and total testosterone were similar within the three groups, with median age 72, 70, and 70 years in Groups 1, 2, and 3, respectively. Three months after the treatment, patients who received Leuprolide 7.5 mg presented significantly lower median total testosterone levels compared with Goserelin 3.6 mg and Leuprolide 3.75 mg (9.5 ng/dL vs. 20.0 ng/dL vs. 30.0 ng/dL, respectively; p = .0072), while those who received Goserelin 3.6 mg presented significantly lower PSA levels compared with Leuprolide 7.5 mg and Leuprolide 3.75 mg (0.67 vs. 1.86 vs. 2.57, respectively; p = .0067). There was no linear correlation between total testosterone and PSA levels. Overall, regarding castration levels of total testosterone, 28.77% of patients did not obtain levels ≤50 ng/dL and 47.80% did not obtain levels ≤20 ng/dL. There was no correlation between total testosterone and PSA kinetics and no equivalence among different pharmacological castrations.

Staphylococcus Aureus Enterotoxins A And B Inhibit Human And Mice Eosinophil Chemotaxis And Adhesion In Vitro.

Staphylococcus aureus aggravates the allergic eosinophilic inflammation. We hypothesized that Staphylococcus aureus-derived enterotoxins directly affect eosinophil functions. Therefore, this study investigated the effects of Staphylococcal enterotoxins A and B (SEA and SEB) on human and mice eosinophil chemotaxis and adhesion in vitro, focusing on p38 MAPK phosphorylation and intracellular Ca(2+) mobilization. Eosinophil chemotaxis was evaluated using a microchemotaxis chamber, whereas adhesion was performed in VCAM-1 and ICAM-1-coated plates. Measurement of p38 MAPK phosphorylation and intracellular Ca(2+) levels were monitored by flow cytometry and fluorogenic calcium-binding dye, respectively. Prior incubation (30 to 240 min) of human blood eosinophils with SEA (0.5 to 3 ng/ml) significantly reduced eotaxin-, PAF- and RANTES-induced chemotaxis (P<0.05). Likewise, SEB (1 ng/ml, 30 min) significantly reduced eotaxin-induced human eosinophil chemotaxis (P<0.05). The reduction of eotaxin-induced human eosinophil chemotaxis by SEA and SEB was prevented by anti-MHC monoclonal antibody (1 μg/ml). In addition, SEA and SEB nearly suppressed the eotaxin-induced human eosinophil adhesion in ICAM-1- and VCAM-1-coated plates. SEA and SEB prevented the increases of p38 MAPK phosphorylation and Ca(2+) levels in eotaxin-activated human eosinophils. In separate protocols, we evaluated the effects of SEA on chemotaxis and adhesion of eosinophils obtained from mice bone marrow. SEA (10 ng/ml) significantly reduced the eotaxin-induced chemotaxis along with cell adhesion to both ICAM-1 and VCAM-1-coated plates (P<0.05). In conclusion, the inhibition by SEA and SEB of eosinophil functions (chemotaxis and adhesion) are associated with reductions of p38 MAPK phosphorylation and intracellular Ca(2+) mobilization.