L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Shear bond strength of resin cements to human dentin

OBJECTIVES: The objectives of this in vitro study were (1) to assess the bond strength of the universal cement RelyX Unicem to dentin and to compare it with three conventional resin cements, (2) to test the influence of aging on their bonding capacity and (3) to test the influence of the operator on bonding quality by performing the same test in two different centers. METHODS: 160 third molars, divided into 80 for tests at the University of Zurich (Z) and 80 for tests at the University of Berne (B), were assigned to 2 x 8 subgroups of 10 teeth each. The specimens were prepared with the corresponding bonding agents and acrylic rods were luted either with RelyX Unicem (U), RelyX ARC (A), Multilink (M) or Panavia 21 (P). All specimens were stored in water for 24h (W) and half of the specimens were subjected to 1500 cycles of thermocycling (5 degrees C and 55 degrees C) (T). Bond strength was measured by means of a shear test. RESULTS: After water storage RelyX Unicem exhibited lowest bond strength (UWZ: 9.2+/-1.6 MPa, UWB: 9.9+/-1.2 MPa, AWZ: 15.3+/-6.0 MPa, AWB: 12.2+/-4.3 MPa, MWZ: 15.6+/-3.3 MPa, MWB: 12.4 MPa+/-2.4, PWZ: 13.4+/-2.9 MPa, PWB: 14.9+/-2.6 MPa). Thermocycling affected the bonding performance of all four cements. However, bond strength of RelyX Unicem was least influenced by thermocycling (UTZ: 9.4+/-2.9 MPa, UTB: 8.6+/-1.3 MPa, ATZ: 11.4+/-6.3 MPa, ATB: 13.3+/-3.7 MPa, MTZ: 15.4+/-3.1 MPa, MTB: 10.3+/-2.4 MPa, PTZ: 11.1+/-2.8 MPa, PTB: 11.3+/-2.8 MPa). SIGNIFICANCE: Although the bond strength of RelyX Unicem to dentin was lower in comparison to RelyX ARC, Multilink and Panavia 21, its bond strength was less sensitive to variations in handling and aging.

Let the Sun Shine In: Open Government and the Public's Right-to-Know in Montana's 1972 Constitution - Mike Meloy "In the Crucible of Change"

Abstract to be posted.

Divergent adaptation of hepatitis C virus genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure

Many hepatitis C virus (HCV) infections worldwide are with the genotype 1 and 3 strains of the virus. Cellular immune responses are known to be important in the containment of HCV genotype 1 infection, and many genotype 1 T cell targets (epitopes) that are presented by host human leukocyte antigens (HLAs) have been identified. In contrast, there is almost no information known about the equivalent responses to genotype 3. Immune escape mechanisms used by HCV include the evolution of viral polymorphisms (adaptations) that abrogate this host-viral interaction. Evidence of HCV adaptation to HLA-restricted immune pressure on HCV can be observed at the population level as viral polymorphisms associated with specific HLA types. To evaluate the escape patterns of HCV genotypes 1 and 3, we assessed the associations between viral polymorphisms and specific HLA types from 187 individuals with genotype 1a and 136 individuals with genotype 3a infection. We identified 51 HLA-associated viral polymorphisms (32 for genotype 1a and 19 for genotype 3a). Of these putative viral adaptation sites, six fell within previously published epitopes. Only two HLA-associated viral polymorphisms were common to both genotypes. In the remaining sites with HLA-associated polymorphisms, there was either complete conservation or no significant HLA association with viral polymorphism in the alternative genotype. This study also highlights the diverse mechanisms by which viral evasion of immune responses may be achieved and the role of genotype variation in these processes. CONCLUSION: There is little overlap in HLA-associated polymorphisms in the nonstructural proteins of HCV for the two genotypes, implying differences in the cellular immune pressures acting on these viruses and different escape profiles. These findings have implications for future therapeutic strategies to combat HCV infection, including vaccine design.

The Netherlands: Darfurnica, Miffy and the right to parody!

VAR

On the Search for an Adequate Scope of the Right to Be Forgotten

During the last decades, the virtual world increasingly gained importance and in this context the enforcement of privacy rights became more and more difficult. An important emanation of this trend is the right to be forgotten enshrining the protection of the data subject’s rights over his/her “own” data. Even though the right to be forgotten has been made part of the proposal for a completely revised Data Protection Regulation and has recently been acknowledged by the Court of Justice of the European Union (“Google/Spain” decision), to date, the discussions about the right and especially its implementation with regard to the fundamental right to freedom of expression have remained rather vague and need to be examined in more depth.

Activation of the lectin pathway of complement in pig-to-human xenotransplantation models

BACKGROUND Natural IgM containing anti-Gal antibodies initiates classic pathway complement activation in xenotransplantation. However, in ischemia-reperfusion injury, IgM also induces lectin pathway activation. The present study was therefore focused on lectin pathway as well as interaction of IgM and mannose-binding lectin (MBL) in pig-to-human xenotransplantation models. METHODS Activation of the different complement pathways was assessed by cell enzyme-linked immunosorbent assay using human serum on wild-type (WT) and α-galactosyl transferase knockout (GalTKO)/hCD46-transgenic porcine aortic endothelial cells (PAEC). Colocalization of MBL/MASP2 with IgM, C3b/c, C4b/c, and C6 was investigated by immunofluorescence in vitro on PAEC and ex vivo in pig leg xenoperfusion with human blood. Influence of IgM on MBL binding to PAEC was tested using IgM depleted/repleted and anti-Gal immunoabsorbed serum. RESULTS Activation of all the three complement pathways was observed in vitro as indicated by IgM, C1q, MBL, and factor Bb deposition on WT PAEC. MBL deposition colocalized with MASP2 (Manders' coefficient [3D] r=0.93), C3b/c (r=0.84), C4b/c (r=0.86), and C6 (r=0.80). IgM colocalized with MBL (r=0.87) and MASP2 (r=0.83). Human IgM led to dose-dependently increased deposition of MBL, C3b/c, and C6 on WT PAEC. Colocalization of MBL with IgM (Pearson's coefficient [2D] rp=0.88), C3b/c (rp=0.82), C4b/c (rp=0.63), and C6 (rp=0.81) was also seen in ex vivo xenoperfusion. Significantly reduced MBL deposition and complement activation was observed on GalTKO/hCD46-PAEC. CONCLUSION Colocalization of MBL/MASP2 with IgM and complement suggests that the lectin pathway is activated by human anti-Gal IgM and may play a pathophysiologic role in pig-to-human xenotransplantation.

Suicide assisted by right-to-die associations: a population based cohort study

Background: In Switzerland, assisted suicide is legal but there is concern that vulnerable or disadvantaged groups are more likely to die in this way than other people. We examined socio-economic factors associated with assisted suicide. Methods: We linked the suicides assisted by right-to-die associations during 2003–08 to a census-based longitudinal study of the Swiss population. We used Cox and logistic regression models to examine associations with gender, age, marital status, education, religion, type of household, urbanization, neighbourhood socio-economic position and other variables. Separate analyses were done for younger (25 to 64 years) and older (65 to 94 years) people. Results: Analyses were based on 5 004 403 Swiss residents and 1301 assisted suicides (439 in the younger and 862 in the older group). In 1093 (84.0%) assisted suicides, an underlying cause was recorded; cancer was the most common cause (508, 46.5%). In both age groups, assisted suicide was more likely in women than in men, those living alone compared with those living with others and in those with no religious affiliation compared with Protestants or Catholics. The rate was also higher in more educated people, in urban compared with rural areas and in neighbourhoods of higher socio-economic position. In older people, assisted suicide was more likely in the divorced compared with the married; in younger people, having children was associated with a lower rate. Conclusions: Assisted suicide in Switzerland was associated with female gender and situations that may indicate greater vulnerability such as living alone or being divorced, but also with higher education and higher socio-economic position.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.