Preclinical research in Rett syndrome: setting the foundation for translational success

In September of 2011, the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the International Rett Syndrome Foundation (IRSF) and the Rett Syndrome Research Trust (RSRT) convened a workshop involving a broad cross-section of basic scientists, clinicians and representatives from the National Institutes of Health (NIH), the US Food and Drug Administration (FDA), the pharmaceutical industry and private foundations to assess the state of the art in animal studies of Rett syndrome (RTT). The aim of the workshop was to identify crucial knowledge gaps and to suggest scientific priorities and best practices for the use of animal models in preclinical evaluation of potential new RTT therapeutics. This review summarizes outcomes from the workshop and extensive follow-up discussions among participants, and includes: (1) a comprehensive summary of the physiological and behavioral phenotypes of RTT mouse models to date, and areas in which further phenotypic analyses are required to enhance the utility of these models for translational studies; (2) discussion of the impact of genetic differences among mouse models, and methodological differences among laboratories, on the expression and analysis, respectively, of phenotypic traits; and (3) definitions of the standards that the community of RTT researchers can implement for rigorous preclinical study design and transparent reporting to ensure that decisions to initiate costly clinical trials are grounded in reliable preclinical data.     

Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex

Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.     

Circulating and airway neutrophils in cystic fibrosis display different TLR expression and responsiveness to interleukin-10

We compared blood neutrophils (PMNs) collected from healthy subjects with PMNs derived from either blood or airways collected from the same cystic fibrosis (CF) patients. When compared to healthy blood PMNs, CF blood PMNs expressed enhanced level of CD64, a marker of neutrophil activation, and lower level of Toll-like receptor-2 (TLR2). CF airway PMNs expressed enhanced level of TLR4. Interleukin-8 (IL-8) production by CF blood PMNs could be enhanced upon addition of lipopolysaccharide or peptidoglycan, and this production was inhibited by recombinant human IL-10. In contrast, CF airway PMNs released spontaneously high level of IL-8 that was neither further enhanced by microbial activators nor inhibited by recombinant human IL-10. The levels of IL-10 receptors were similar in all types of neutrophils. These data further demonstrate that circulating PMNs from CF patients display a distinct pattern of surface markers, including TLRs, as compared to PMNs from healthy donors, and that airways PMNs from CF patients are primed and resistant to anti-inflammatory signals delivered by IL-10.     

Neutrophils in cystic fibrosis display a distinct gene expression pattern

We compared gene expression in blood neutrophils (polymorphonuclear leukocytes, or PMNs) collected from healthy subjects with those of cystic fibrosis (CF) patients devoid of bacterial colonization. Macroarray analysis of 1050 genes revealed upregulation of 62 genes and downregulation expression of 27 genes in CF blood PMNs. Among upregulated genes were those coding for vitronectin, some chemokines (particularly CCL17 and CCL18), some interleukin (IL) receptors (IL-3, IL-8, IL-10, IL-12), all three colony-stimulating factors (G-, M-, GM-CSF), numerous genes coding for molecules involved in signal transduction, and a few genes under the control of gamma-interferon. In contrast, none of the genes coding for adhesion molecules were modulated. The upregulation of six genes in CF PMNs (coding for thrombospondin-1, G-CSF, CXCL10, CCL17, IKKvarepsilon, IL-10Ra) was further confirmed by qPCR. In addition, the increased presence of G-CSF, CCL17, and CXCL10 was confirmed by ELISA in supernatants of neutrophils from CF patients. When comparison was performed between blood and airway PMNs of CF patients, there was a limited difference in terms of gene expression. Only the mRNA expression of amphiregulin and tumor necrosis factor (TNF) receptor p55 were significantly higher in airway PMNs. The presence of amphiregulin was confirmed by ELISA in the sputum of CF patients, suggesting for the first time a role of amphiregulin in cystic fibrosis. Altogether, this study clearly demonstrates that blood PMNs from CF patients display a profound modification of gene expression profile associated with the disease, suggesting a state of activation of these cells.     

Effect of Precursor Solution Aging on the Crystallinity and Photovoltaic Performance of Perovskite Solar Cells

Perovskite materials due to their exceptional photophysical properties are beginning to dominate the field of thin‐film optoelectronic devices. However, one of the primary challenges is the processing‐dependent variability in the properties, thus making it imperative to understand the origin of such variations. Here, it is discovered that the precursor solution aging time before it is cast into a thin film, is a subtle but a very important factor that dramatically affects the overall thin‐film formation and crystallinity and therein factors such as grain growth, phase purity, surface uniformity, trap state density, and overall solar cell performance. It is shown that progressive aging of the precursor promotes efficient formation of larger seeds after the fast nucleation of a large density of small seeds. The hot‐casting method then leads to the growth of large grains in uniform thin‐films with excellent crystallinity validated using scanning microscopy images and X‐ray diffraction patterns. The high‐quality films cast from aged solution is ideal for thin‐film photovoltaic device fabrication with reduced shunt current and good charge transport. This observation is a significant step toward achieving highly crystalline thin‐films with reliability in device performance and establishes the subtle but dramatic effect of solution aging before fabricating perovskite thin‐films.     

Running demands and activity profile of men’s rugby sevens: a tournament scenario

This study profiled the changes in running performances and collisions within a Rugby sevens tournament. Sixteen male players were equipped with global positioning system units while competing at the 2015 and 2016 Asia Rugby Sevens series held in Colombo and Hong Kong, respectively. Both tournaments consisted of 4 matches each, and were played over 2 days (i.e., 2 matches/day). Total distance (TD) covered increased in match 3 compared with matches 1 (19 ± 19%; p < 0.001) and 2 (16 ± 11%; p = 0.001), whilst a decrease in TD in match 4 compared with match 3 (8 ± 9%; p = 0.019) was observed. Distances covered within 6.1–12 km·h^-1 and 12.1–14 km·h^-1 speed bands were generally higher in matches 3 and/or 4 when compared with match 1 and/or 2 (p < 0.05). Frequency of entries into 14.1–18 km·h^-1 speed zone was decreased in match 4 compared with match 3 (45 ± 41%; p = 0.009), whilst incidences of heavy, very heavy and severe collisions were generally higher in matches 3 or 4 compared with matches 1 or 2 (p < 0.05). In conclusion, while some decrements in the final match were evident, running performance were generally maintained throughout despite the competitive and congested nature of Rugby Sevens tournaments     

Biomechanical remodeling of the microenvironment by stromal caveolin-1 favors tumor invasion and metastasis

Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.