WW Domains of the Yes-Kinase-Associated-Protein (YAP) Transcriptional Regulator Behave as Independent Units with Different Binding Preferences for PPxY Motif-Containing Ligands

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.This work was supported by the Spanish Ministry of Education and Science [grant BIO2009-13261-CO2], the Spanish Ministry of Economy and Competitivity [grant BIO2012-39922-CO2] including FEDER (European Funds for Regional Development) funds and the Governement of Andalusia [grant CVI-5915]. Marius Sudol was supported by PA Breast Cancer Coalition Grants (#60707 and #920093) plus the Geisinger Clinic

Properties of Graphene: A Theoretical Perspective

In this review, we provide an in-depth description of the physics of monolayer and bilayer graphene from a theorist's perspective. We discuss the physical properties of graphene in an external magnetic field, reflecting the chiral nature of the quasiparticles near the Dirac point with a Landau level at zero energy. We address the unique integer quantum Hall effects, the role of electron correlations, and the recent observation of the fractional quantum Hall effect in the monolayer graphene. The quantum Hall effect in bilayer graphene is fundamentally different from that of a monolayer, reflecting the unique band structure of this system. The theory of transport in the absence of an external magnetic field is discussed in detail, along with the role of disorder studied in various theoretical models. We highlight the differences and similarities between monolayer and bilayer graphene, and focus on thermodynamic properties such as the compressibility, the plasmon spectra, the weak localization correction, quantum Hall effect, and optical properties. Confinement of electrons in graphene is nontrivial due to Klein tunneling. We review various theoretical and experimental studies of quantum confined structures made from graphene. The band structure of graphene nanoribbons and the role of the sublattice symmetry, edge geometry and the size of the nanoribbon on the electronic and magnetic properties are very active areas of research, and a detailed review of these topics is presented. Also, the effects of substrate interactions, adsorbed atoms, lattice defects and doping on the band structure of finite-sized graphene systems are discussed. We also include a brief description of graphane -- gapped material obtained from graphene by attaching hydrogen atoms to each carbon atom in the lattice.Comment: 189 pages. submitted in Advances in Physic

Rapid method for determination of DNA repair capacity in human peripheral blood lymphocytes amongst smokers

<p>Abstract</p> <p>Background</p> <p>DNA repair capacity is an important determinant of susceptibility to cancer. The hOGG1 enzyme is crucial for repairing the 8-oxoguanine lesion that occurs either as a byproduct of oxidative metabolism or as a result of exogenous sources such as exposure to cigarette smoke. It has been previously reported that smokers with low hOGG1 activity had significantly higher risk of developing lung cancer as compared to smokers with high hOGG1 activity.</p> <p>Methods</p> <p>In the current study we elucidate the association between plasma levels of 8-OHdG and the OGG1 repair capacity. We used the commercially available 8-OHdG ELISA (enzyme-linked immunosorbent assay), the Comet assay/FLARE hOGG1 (Fragment Length Analysis by Repair Enzymes) assay for quantification of the levels of 8-OHdG and measured the constitutive, induced and unrepaired residual damage, respectively. We compared the DNA repair capacity in peripheral blood lymphocytes following H₂O₂exposure in 30 lung cancer patients, 30 non-, 30 former and 30 current smoker controls matched by age and gender.</p> <p>Results</p> <p>Our results show that lung cancer cases and current smoker controls have similar levels of 8-OHdG lesions that are significantly higher compared to the non-smokers controls. However, lung cancer cases showed significantly poorer repair capacity compared to all controls tested, including the current smokers controls. After adjustment for age, gender and family history of smoking-related cancer using linear regression, we observed a 5-fold increase in risk of lung cancer associated with high levels of residual damage/reduced repair capacity. Reduced OGG1 activity could be expected to be a risk factor in other smoking-related cancers.</p> <p>Conclusion</p> <p>Our study shows that the Comet/FLARE assay is a relatively rapid and useful method for determination of DNA repair capacity. Using this assay we could identify individuals with high levels of residual damage and hence poor repair capacity who would be good candidates for intensive follow-up and screening.</p

Association of self-reported physical activity patterns and socio-demographic factors among normal-weight and overweight Japanese men

<p>Abstract</p> <p>Background</p> <p>It is still not known whether overweight men have different patterns and socio-demographic correlates of self-reported physical activity (PA) compared with normal-weight men. Thus, this study examined the perceived PA patterns and associated socio-demographic factors among normal-weight and overweight Japanese men.</p> <p>Methods</p> <p>Data were analyzed for 1,420 men (aged 44.48.3years) who responded to an Internet-based cross-sectional survey relating to socio-demographic variables, BMI status, and a short version of the International Physical Activity Questionnaire. Mann-Whitney, chi-square, and binary logistic regression analyses were employed.</p> <p>Results</p> <p>Normal-weight men were significantly more likely to attain 150 minutes per week of moderate-to-vigorous PA than overweight men (26.6% vs. 21.3%; <it>p</it>=0.035), whereas there were no significant proportional differences in total PA and walking between the two BMI subgroups. With PA, a significant interaction was observed between BMI status and household income (<it>p</it>=0.004 for total PA; <it>p</it>=0.02 for walking). In the subgroup analyses, having a lower household income (odds ratio, 0.63; 95% confidence interval, 0.41-0.96) was negatively associated with attaining 150 minutes of walking per week among normal-weight men. No significant associations between household income and attaining 150 minutes per week of total PA and walking were found among overweight men.</p> <p>Conclusions</p> <p>The results revealed that patterns and socio-demographic correlates of self-reported PA in overweight men are different from those in normal-weight men. This finding suggests the necessity of developing specific strategies for PA intervention among overweight men. Socio-demographic correlates of PA may be more important for normal-weight than overweight men.</p

Alpha-1-acid glycoprotein as potential biomarker for alpha-fetoprotein-low hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>The outcome of patients with hepatocellular carcinoma (HCC) remains poor because of late diagnosis. We determined the performances of α -1-acid glycoprotein (AAG) and des-γ-carboxy prothrombin (DCP) for the diagnosis of HCC, especially for α-fetoprotein (AFP)-low HCC.</p> <p>Methods</p> <p>Of the 220 patients included in this retrospective study, 124 had HCC, and 61 (49%) of these were AFP-low HCC (AFP ≤ 20 ng/mL). The remaining 96 patients, including 49 with chronic hepatitis B or C and 47 with cirrhosis, were considered as control. Plasma AAG was analyzed using high performance liquid chromatography (HPLC) and confirmed using Western blot technique.</p> <p>Results</p> <p>When all patients with HCC were evaluated, the area under receiver operating characteristic (ROC) curves for AAG (0.94, 95% CI: 0.91-0.97) and DCP (0.92, 95% CI: 0.88-0.95) were similar (<it>P </it>= 0.40). AAG had better area under ROC curve (0.96, 95% CI: 0.94-0.99) than DCP (0.87, 95% CI: 0.81-0.93) for AFP-low HCC (<it>P </it>< 0.05). At the specificity 95%, the sensitivity of AAG was higher in AFP-low HCC than in AFP-high HCC (82% and 62%, respectively). In contrast, higher sensitivity was obtained from DCP in discriminating HCC patients with low AFP than that in high AFP (57% and 90%, respectively).</p> <p>Conclusion</p> <p>Our cross-sectional study showed that AAG was better performance in diagnosing HCC patients with low AFP, while DCP did better in those with high AFP.</p

Bacterial Delivery of Nuclear Proteins into Pluripotent and Differentiated Cells

Numerous Gram negative pathogens possess a type III secretion system (T3SS) which allows them to inject virulent proteins directly into the eukaryotic cell cytoplasm. Injection of these proteins is dependent on a variable secretion signal sequence. In this study, we utilized the N-terminal secretion signal sequence of Pseudomonas aeruginosa exotoxin ExoS to translocate Cre recombinase containing a nuclear localization sequence (Cre-NLS). Transient exposure of human sarcoma cell line, containing Cre-dependent lacZ reporter, resulted in efficient recombination in the host chromosome, indicating that the bacterially delivered protein was not only efficiently localized to the nucleus but also retained its biological function. Using this system, we also illustrate the ability of P. aeruginosa to infect mouse embryonic stem cells (mESC) and the susceptibility of these cells to bacterially delivered Cre-NLS. A single two-hour infection caused as high as 30% of the mESC reporter cells to undergo loxP mediated chromosomal DNA recombination. A simple antibiotic treatment completely eliminated the bacterial cells following the delivery, while the use of an engineered mutant strain greatly reduced cytotoxicity. Utility of the system was demonstrated by delivery of the Cre-NLS to induced pluripotent stem cells to excise the floxed oncogenic nuclear reprogramming cassette. These results validate the use of T3SS for the delivery of transcription factors for the purpose of cellular reprogramming

Trypanosoma vivax Infections: Pushing Ahead with Mouse Models for the Study of Nagana. II. Immunobiological Dysfunctions

Trypanosoma vivax is the main species involved in trypanosomosis, but very little is known about the immunobiology of the infective process caused by this parasite. Recently we undertook to further characterize the main parasitological, haematological and pathological characteristics of mouse models of T. vivax infection and noted severe anemia and thrombocytopenia coincident with rising parasitemia. To gain more insight into the organism's immunobiology, we studied lymphocyte populations in central (bone marrow) and peripherical (spleen and blood) tissues following mouse infection with T. vivax and showed that the immune system apparatus is affected both quantitatively and qualitatively. More precisely, after an initial increase that primarily involves CD4+ T cells and macrophages, the number of splenic B cells decreases in a step-wise manner. Our results show that while infection triggers the activation and proliferation of Hematopoietic Stem Cells, Granulocyte-Monocyte, Common Myeloid and Megacaryocyte Erythrocyte progenitors decrease in number in the course of the infection. An in-depth analysis of B-cell progenitors also indicated that maturation of pro-B into pre-B precursors seems to be compromised. This interferes with the mature B cell dynamics and renewal in the periphery. Altogether, our results show that T. vivax induces profound immunological alterations in myeloid and lymphoid progenitors which may prevent adequate control of T. vivax trypanosomosis

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9(hi), SSEA-1(−) Cells in Embryonic Stem Cell-Derived Embryoid Bodies

BACKGROUND: Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. METHODOLOGY/PRINCIPAL FINDINGS: We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E- RoSH lines have similar gene expression profiles (r(2) = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi), SSEA-1(−) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(−) cells that constituted 1%–10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 0.95) and a propensity to differentiate into endothelial-like cells. CONCLUSIONS: By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs

VHA-19 Is Essential in Caenorhabditis elegans Oocytes for Embryogenesis and Is Involved in Trafficking in Oocytes

There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described