Differences in wild-type– and R338L-tenase complex formation are at the root of R338L-factor IX assay discrepancies

Adeno-associated virus (AAV) gene therapy has the potential to functionally cure hemophilia B by restoring factor (F)IX concentrations into the normal range. Next-generation AAV therapies express a naturally occurring gain-of-function FIX variant, FIX-Padua (R338L-FIX), that increases FIX activity (FIX:C) by approximately eightfold compared with wild-type FIX (FIX-WT). Previous studies have shown that R338L-FIX activity varies dramatically across different clinical FIX:C assays, which complicates the monitoring and management of patients. To better understand mechanisms that contribute to R338L-FIX assay discrepancies, we characterized the performance of R338L-FIX in 13 1-stage clotting assays (OSAs) and 2 chromogenic substrate assays (CSAs) in a global field study. This study produced the largest R338L-FIX assay dataset to date and confirmed that clinical FIX:C assay results vary over threefold. Both phospholipid and activating reagents play a role in OSA discrepancies. CSA generated the most divergent FIX:C results. Manipulation of FIX:C CSA kits demonstrated that specific activity gains for R338L-FIX were most profound at lower FIX:C concentrations and that these effects were enhanced during the early phases of FXa generation. Supplementing FX into CSA had the effect of dampening FIX-WT activity relative to R338L-FIX activity, suggesting that FX impairs WT tenase formation to a greater extent than R338L-FIX tenase. Our data describe the scale of R338L-FIX assay discrepancies and provide insights into the causative mechanisms that will help establish best practices for the measurement of R338LFIX activity in patients after gene therapy

Vesicular Egress of Non-Enveloped Lytic Parvoviruses Depends on Gelsolin Functioning

The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of “actin patches.” This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIα, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process

Measurements of cross-shore sand fluxes on a macrotidal pocket beach (Saint-Georges Beach, Atlantic Coast, SW France)

Analisis del trasporto cross-shore nella zona costier

Winter Sandy Protections of the Northern Adriatic Coast against Flooding: Preliminary Results

Coastal erosion induces a constant and often permanent loss of territory. This phenomenon is more alarming when assosiated with the lowering of the hinterland territory because of the subsidence. This trend sets the conditions for more frequent and vast floodings due to sea storm and high water events. Such floodings not only bring remarkable uneasiness to people and local economies but, sometimes, they can be transformed in more serious catastrophe, as during the inundation of Venice in 1966. Fortunately, because of the weather-marine and geomorphological conditions of the coast of Emilia-Romagna, in the northern Adriatic sea (Italy) floodings provoke damages principally to infrastructures and human activities. In order to reduce these risks, temporary sandy levees are constructed during the winter period by the owners of the infrastructures: the material used is usually taken from the lower part of the beach and deposited without following a suitable procedure. If these morphological variations are not accurately evaluated, they may induce changes on the beach and increase the actual erosion. In order to reduce such phenomenon and improve the performance of these temporary interventions, a study has been conducte which aims to determine the sea storm impact on the sand levee and to evaluate the most appropriate constructive modalities and the possibility to use sand coming from the cleaning of the beach during the summer period, which is today accumulated in dumps. A methodology based on the use of the SBEACH (Storm-induced BEAch CHange) software is presented. First results are encouraging. More simulation will be performed in order to validate the methodology

Mixing depth experiments on an estuarine beach; St. Georges Beach, Gironde (France)

Mixing depth experiments were carried out on the dissipative St. Georges Beach (Gironde Estuary) in September 1999 (SWAMGIR 2) and May 2000 (SWAMGIR 4). The mixing depths were found to reach maximum values of about 0.15 m during SWAMGIR 2, and 0.1 m during SWAMGIR 4. The analysis of results compares measured data with data calculated using the formula proposed by FERREIRA et al. (2000). Two approaches were used: a) an averaged approach, using global slope and breaking wave height at all points, and b) a non-averaged approach, considering varying breaking wave heights and slopes across the beach. Comparisons show that in general the formula works well for both approaches with calculated results within the magnitude of measured values. However, while the non-averaged approach produced significantly better results on the low-tide terrace, the standard deviation of the difference between calculated and measured values was much higher

NEGOTIATING A RESPONSE TO CLIMATE CHANGE: ROLE OF BIOLOGICAL EMISSIONS

Researchers have attributed 15 percent of global methane releases to emissions of methane from enteric fermentation in animals (wild and domestic). Bovines contribute approximately two-thirds of this amount. Since methane is a potent greenhouse gas, this source frequently is a target for emission reductions. However, the existing literature overstates the importance of bovine methane as a greenhouse gas by as much as 800 percent. Estimates to date have focused solely on gas emissions, ignoring the biological and chemical cycling that removes carbon from the atmosphere. The analysis presented here demonstrates the importance of these cycles in assessing the overall greenhouse effect of biological methane sources such as rice production, termites, and bovine animals. Ignoring this cycling results in overemphasizing the role of developing countries' total contributions to climate change. In economic terms, the analysis shows that reducing CO2 emissions from energy use in industrialized countries is more efficient than reducing net greenhouse methane from animal sources. Copyright 1992 Western Economic Association International.

Safe, long-term hepatic expression of anti-HCV shRNA in a nonhuman primate model

The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a “single-shot ” therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection