Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.     

Regulation of Ncx1 expression. Identification of regulatory elements mediating cardiac-specific expression and up-regulation

The Na+-Ca2+ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.     

C-terminal PAL motif of presenilin and presenilin homologues required for normal active site conformation

The Alzheimer's disease-associated beta-amyloid peptide is produced through cleavage of amyloid precursor protein by beta-secretase and gamma-secretase. gamma-Secretase is a complex containing presenilin (PS) as the catalytic component and three essential cofactors: Nicastrin, anterior pharynx defective (APH-1) and presenilin enhancer-2 (PEN-2). PS and signal peptide peptidase (SPP) define a novel family of aspartyl proteases that cleave substrates within the transmembrane domain presumptively using two membrane-embedded aspartic acid residues for catalysis. Apart from the two aspartate-containing active site motifs, the only other region that is conserved between PS and SPP is a PAL sequence at the C-terminus. Although it has been well documented that this motif is essential for gamma-secretase activity, the mechanism underlying such a critical role is not understood. Here we show that mutations in this motif affect the conformation of the active site of gamma-secretase resulting in a complete loss of PS binding to a gamma-secretase transition state analog inhibitor, Merck C. Analogous mutations in SPP significantly inhibit its enzymatic activity. Furthermore, these mutations also abolish SPP binding to Merck C, indicating that SPP and gamma-secretase share a similar active site conformation, which is dependent on the PAL motif. Exploring the amino acid requirements within this motif reveals a very small side chain requirement, which is conserved during evolution. Together, these observations strongly support the hypothesis that the PAL motif contributes to the active site conformation of gamma-secretase and of SPP.     

Cannabinoid signalling

After their discovery, the two known cannabinoid receptors, CB(1) and CB(2), have been the focus of research into the cellular signalling mechanisms of cannabinoids. The initial assessment, mainly derived from expression studies, was that cannabinoids, via G(i/o) proteins, negatively modulate cyclic AMP levels, and activate inward rectifying K(+) channels. Recent findings have complicated this assessment on different levels: (1) cannabinoids include a wide range of compounds with varying profiles of affinity and efficacy at the known CB receptors, and these profiles do not necessarily match their biological activity; (2) CB receptors appear to be intrinsically active and possibly coupled to more than one type of G protein; (3) CB receptor signalling mechanisms are diverse and dependent on the system studied; (4) cannabinoids have other targets than CB receptors. The aim of this mini review is to discuss the current literature regarding CB receptor signalling pathways. These include regulation of adenylyl cyclase, MAP kinase, intracellular Ca(2+), and ion channels. In addition, actions of cannabinoids that are not mediated by CB(1) or CB(2) receptors are discussed.     

The Walking Behaviour of Pedestrian Social Groups and Its Impact on Crowd Dynamics

Human crowd motion is mainly driven by self-organized processes based on local interactions among pedestrians. While most studies of crowd behaviour consider only interactions among isolated individuals, it turns out that up to 70% of people in a crowd are actually moving in groups, such as friends, couples, or families walking together. These groups constitute medium-scale aggregated structures and their impact on crowd dynamics is still largely unknown. In this work, we analyze the motion of approximately 1500 pedestrian groups under natural condition, and show that social interactions among group members generate typical group walking patterns that influence crowd dynamics. At low density, group members tend to walk side by side, forming a line perpendicular to the walking direction. As the density increases, however, the linear walking formation is bent forward, turning it into a V-like pattern. These spatial patterns can be well described by a model based on social communication between group members. We show that the V-like walking pattern facilitates social interactions within the group, but reduces the flow because of its “non-aerodynamic” shape. Therefore, when crowd density increases, the group organization results from a trade-off between walking faster and facilitating social exchange. These insights demonstrate that crowd dynamics is not only determined by physical constraints induced by other pedestrians and the environment, but also significantly by communicative, social interactions among individuals.     

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.     

Photosynthetic efficiency of Chlorella sorokiniana in a turbulently mixed short light‐path photobioreactor

To be able to study the effect of mixing as well as any other parameter on productivity of algal cultures, we designed a lab‐scale photobioreactor in which a short light path (SLP) of (12 mm) is combined with controlled mixing and aeration. Mixing is provided by rotating an inner tube in the cylindrical cultivation vessel creating Taylor vortex flow and as such mixing can be uncoupled from aeration. Gas exchange is monitored on‐line to gain insight in growth and productivity. The maximal productivity, hence photosynthetic efficiency, of Chlorella sorokiniana cultures at high light intensities (1,500 μmol m^?1 s^?1) was investigated in this Taylor vortex flow SLP photobioreactor. We performed duplicate batch experiments at three different mixing rates: 70, 110, and 140 rpm, all in the turbulent Taylor vortex flow regime. For the mixing rate of 140 rpm, we calculated a quantum requirement for oxygen evolution of 21.2 mol PAR photons per mol O₂ and a yield of biomass on light energy of 0.8 g biomass per mol PAR photons. The maximal photosynthetic efficiency was found at relatively low biomass densities (2.3 g L^?1) at which light was just attenuated before reaching the rear of the culture. When increasing the mixing rate twofold, we only found a small increase in productivity. On the basis of these results, we conclude that the maximal productivity and photosynthetic efficiency for C. sorokiniana can be found at that biomass concentration where no significant dark zone can develop and that the influence of mixing‐induced light/dark fluctuations is marginal. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Power consumption and maximum energy dissipation in a milliliter‐scale bioreactor

Mean power consumption and maximum local energy dissipation were measured as function of operating conditions of a milliliter‐scale stirred tank bioreactor (V = 12 mL) with a gas‐inducing impeller. A standard laboratory‐scale stirred tank bioreactor (V = 1,200 mL) with Rushton turbines was used as reference. The measured power characteristics (Newton number as function of Reynolds number) were the same on both scales. The changeover between laminar and turbulent flow regime was observed at a Reynolds number of 3,000 with the gas‐inducing stirrer on a milliliter‐scale. The Newton number (power number) in the turbulent flow regime was 3.3 on a milliliter‐scale, which is close to values reported for six‐blade Rushton turbines of standard bioreactors. Maximum local energy dissipation (ε_max) was measured using a clay/polymer flocculation system. The maximum local energy dissipation in the milliliter‐scale stirred tank bioreactor was reduced compared with the laboratory‐scale stirred tank at the same mean power input per unit mass (ε_ø), yielding ε_max/ε_ø ≈ 10 compared with ε_max/ε_ø ≈ 16. Hence, the milliliter‐scale stirred tank reactor distributes power more uniformly in the reaction medium. These results are in good agreement with literature data, where a decreasing ε_max/ε_ø with increasing ratio of impeller diameter to reactor diameter is found (d/D = 0.7 compared with d/D = 0.4). Based on these data, impeller speeds can now be easily adjusted to achieve the same maximum local energy dissipation at different scales. This enables a more reliable and robust scale‐up of bioprocesses from milliliter‐scale to liter‐scale reactors. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Pathogen proteomes during infection: A basis for infection research and novel control strategies

Infectious diseases cause tremendous mortality and morbidity worldwide. Rising antimicrobial resistance and the lack of new drugs cause an increasingly alarming crisis in infectious disease control. New system-level approaches are likely to help understand complex host/pathogen interactions as a basis for rational development of novel antibiotics and vaccines. Proteome analysis of pathogens in infected tissues comprehensively reveals functionally relevant pathogen activities during infection. It also highlights potential targets for antimicrobial chemotherapy as well as promising antigens for vaccination. Integration of these data with complementary large-scale data helps to further prioritize candidates for in-depth experimental analysis. Here, I discuss some of these approaches with a special emphasis on the model pathogen Salmonella.     

Response of Pseudomonas putida KT2440 to phenol at the level of membrane proteome

This study led to the extension and refinement of our current model for the global response of Pseudomonas putida KT2440 to phenol by getting insights into the adaptive response mechanisms involving the membrane proteome. A two-dimensional gel electrophoresis based protocol was optimized to allow the quantitative comparison of membrane proteins, by combining inner and outer membrane fractionation with membrane protein solubilization using the detergent dodecylmaltoside. Following phenol exposure, a coordinate increased content of protein subunits of known or putative solvent efflux pump systems (e.g. TtgA, TtgC, Ttg2A, Ttg2C, and PP_1516-7) and a decreased content of porins OprB, OprF, OprG and OprQ was registered, consistent with an adaptive response to reduce phenol intracellular concentration. This adaptive response may in part be mediated by post-translational modifications, as suggested by the relative content of the multiple forms identified for a few porins and efflux pump subunits. Results also suggest the important role of protein chaperones, of cell envelope and cell surface and of a more active respiratory chain in the response to phenol. All these mechanistic insights may be extended to Pseudomonas adaptation to solvents, of possible impact in biodegradation, bioremediation and biocatalysis.