Alirocumab,a Therapeutic Human Antibody to PCSK9, Does Not Affect CD81 Levels or Hepatitis C Virus Entry and Replication into Hepatocytes

BackgroundProprotein convertase subtilisin/kexin type 9 (PSCK9) is secreted mainly from the liver and binds to the low-density lipoprotein receptor (LDLR), reducing LDLR availability and thus resulting in an increase in LDL-cholesterol. While the LDLR has been implicated in the cell entry process of the hepatitis C virus (HCV), overexpression of an artificial non-secreted, cell membrane-bound form of PCSK9 has also been shown to reduce surface expression of CD81, a major component of the HCV entry complex, leading to concerns that pharmacological inhibition of PCSK9 may increase susceptibility to HCV infection by increasing either CD81 or LDLR availability. Here, we evaluated effects of PCSK9 and PCSK9 blockade on CD81 levels and HCV entry with a physiologically relevant model using native secreted PCSK9 and a monoclonal antibody to PCSK9, alirocumab.ConclusionThese results suggest that inhibition of PCSK9 with alirocumab has no effect on CD81 and does not result in increased susceptibility to HCV entry.     

Spatially balanced sampling and ground‐level imagery for vegetation monitoring on reclaimed well pads

Land reclamation associated with natural gas development has become increasingly important to mitigate land surface disturbance in western North America. Since well pads occur on sites with multiple land use and ownership, the progress and outcomes of these efforts are of interest to multiple stakeholders including industry, practitioners and consultants, regulatory agents, private landowners, and the scientific community. Reclamation success criteria often vary within, and among, government agencies and across land ownership type. Typically, reclamation success of a well pad is judged by comparing vegetation cover from a single transect on the pad to a single transect in an adjacent reference site and data are collected by a large number of technicians with various field monitoring skills. We utilized “SamplePoint” image analysis software and a spatially balanced sampling design, called balanced acceptance sampling, to demonstrate how spatially explicit quantitative data can be used to determine if sites are meeting various reclamation success criteria and used chi‐square tests to show how sites in vegetation percent cover differ from a statistical standpoint. This method collects field data faster than traditional methods. We demonstrate how quantitative and spatially explicit data can be utilized by multiple stakeholders, how it can improve upon current reference site selection, how it can satisfy reclamation monitoring requirements for multiple regulatory agencies, how it may help improve future seed mix selection, and discuss how it may reduce costs for operations responsible for reclamation and how it may reduce observer bias.     

Passive and facilitated transport in nuclear pore complexes is largely uncoupled

Nuclear pore complexes provide the sole gateway for the exchange of material between nucleus and cytoplasm of interphase eukaryotic cells. They support two modes of transport: passive diffusion of ions, metabolites, and intermediate-sized macromolecules and facilitated, receptor-mediated translocation of proteins, RNA, and ribonucleoprotein complexes. It is generally assumed that both modes of transport occur through a single diffusion channel located within the central pore of the nuclear pore complex. To test this hypothesis, we studied the mutual effects between transporting molecules utilizing either the same or different modes of translocation. We find that the two modes of transport do not interfere with each other, but molecules utilizing a particular mode of transport do hinder motion of others utilizing the same pathway. We therefore conclude that the two modes of transport are largely segregated.     

Three-dimensional ultrastructure and quantitative analysis of the human Sertoli cell nucleolus

The nucleolus of the human Sertoli cell displays a spontaneous segregation of its components and has only one or 2 large fibrillar centers. The 3-dimensional reconstruction and quantitative analysis of its components was undertaken using a Quantimet 900 image analysis system in order to define the spatial relationships between the dense fibrillar component and the fibrillar center and especially to investigate whether threads of dense fibrillar component exist independently, without being linked to a fibrillar center. Our 3D reconstructions demonstrated that the dense fibrillar threads or sheets were never independent of fibrillar centers. These structures belonged to a continuous network that joined the layer of dense fibrils surrounding the fibrillar center. When the nucleolus contained 2 different-sized fibrillar centers, quantitative analysis showed that there was a proportional relationship between the volume of the dense fibrillar component and the volume of the fibrillar center. These data, compared with those previously obtained by means of autoradiographic techniques, suggest that the rDNA-containing chromatin passes through the fibrillar center and unwinds from there into the dense fibrillar component.     

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.     

Genetic engineering of brewing yeast to reduce the content of ethanol in beer

The GPD1 gene encoding the glycerol-3-phosphate dehydrogenase was overexpressed in an industrial lager brewing yeast (Saccharomyces cerevisiae ssp. carlsbergensis) to reduce the content of ethanol in beer. The amount of glycerol produced by the GPD1-overexpressing yeast in fermentation experiments simulating brewing conditions was increased 5.6 times and ethanol was decreased by 18% when compared to the wild-type. Overexpression of GPD1 does not affect the consumption of wort sugars. Only minor changes in the concentration of higher alcohols, esters and fatty acids could be observed in beer produced by the GPD1-overexpressing brewing yeast. However, the concentrations of several other by-products, particularly acetoin, diacetyl and acetaldehyde, were considerably increased.     

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.