Transcriptomic analysis of responses to exudates reveal genes required for rhizosphere competence of the endophyte Azoarcus sp. strain BH72

Endophytic colonization is a very complex process which is not yet completely understood. Molecules exuded by the plants may act as signals which influence the ability of the microbe to colonize the host or survive in the rhizosphere. Here we used the whole genome microarray approach to investigate the response of the diazotrophic model endophyte, Azoarcus sp. strain BH72, to exudates of O.?sativa cv. Nipponbare in order to identify differentially regulated genes. On exposure to exudates, an overall expression of 4.4% of the 3992 protein coding genes of Azoarcus sp. strain BH72 was altered, out of which 2.4% was upregulated and 2.0% was downregulated. Genes with modulated expression included a few whose involvement in plant-microbe interaction had already been established, whereas a large fraction comprised of genes encoding proteins with putative or unknown functions. Mutational analysis of several differentially regulated genes like those encoding a minor pilin PilX, signal transduction proteins containing GGDEF domains and a serine-threonine kinase as a putative component of the type IV secretion system (T6SS), revealed their role in host colonization. Our data suggest that strain BH72 may be primed for the endophytic lifestyle by exudates, as the expression of bacterial genes relevant for endophytic colonization of roots is induced by root exudates.     

Measles Virus Glycoprotein-Based Lentiviral Targeting Vectors That Avoid Neutralizing Antibodies

Lentiviral vectors (LVs) are potent gene transfer vehicles frequently applied in research and recently also in clinical trials. Retargeting LV entry to cell types of interest is a key issue to improve gene transfer safety and efficacy. Recently, we have developed a targeting method for LVs by incorporating engineered measles virus (MV) glycoproteins, the hemagglutinin (H), responsible for receptor recognition, and the fusion protein into their envelope. The H protein displays a single-chain antibody (scFv) specific for the target receptor and is ablated for recognition of the MV receptors CD46 and SLAM by point mutations in its ectodomain. A potential hindrance to systemic administration in humans is pre-existing MV-specific immunity due to vaccination or natural infection. We compared transduction of targeting vectors and non-targeting vectors pseudotyped with MV glycoproteins unmodified in their ectodomains (MV-LV) in presence of α-MV antibody-positive human plasma. At plasma dilution 1∶160 MV-LV was almost completely neutralized, whereas targeting vectors showed relative transduction efficiencies from 60% to 90%. Furthermore, at plasma dilution 1∶80 an at least 4-times higher multiplicity of infection (MOI) of MV-LV had to be applied to obtain similar transduction efficiencies as with targeting vectors. Also when the vectors were normalized to their p24 values, targeting vectors showed partial protection against α-MV antibodies in human plasma. Furthermore, the monoclonal neutralizing antibody K71 with a putative epitope close to the receptor binding sites of H, did not neutralize the targeting vectors, but did neutralize MV-LV. The observed escape from neutralization may be due to the point mutations in the H ectodomain that might have destroyed antibody binding sites. Furthermore, scFv mediated cell entry via the target receptor may proceed in presence of α-MV antibodies interfering with entry via the natural MV receptors. These results are promising for in vivo applications of targeting vectors in humans.     

Modified "4 + 1" mixed ligand technetium-labeled fatty acids for myocardial imaging: evaluation of myocardial uptake and biodistribution

Our group previously synthesized 99m Tc-labeled fatty acids suitable for myocardial metabolism and flow imaging. In this set of experiments, 29 new analogues were synthesized according to the "4 + 1" mixed ligand approach with some specific differences. Conventional "4 + 1" 99m Tc-fatty acids are built in the sequence: Tc-chelate, alkyl chain, and carboxylic group. We developed compounds following a new design with the sequence: carboxylic group, alkyl chain, Tc-chelate, and lipophilic tail. Therefore, the 99m Tc-chelate was transferred to a more central position of the compound, aiming toward an improved myocardial profile and an accelerated liver clearance. In this context, several functional groups incorporated in the lipophilic tail section were tested to evaluate their influence on the compound's character. In addition to biodistribution studies in vivo, the myocardial first-pass extraction of the compounds was tested in an isolated Langendorff rat heart model. A satisfactory myocardial uptake of up to 20% of the injected dose (% ID) in the perfused heart and a fast liver clearance in vivo with only 0.29% ID/g at 60 min postinjection demonstrate that the induced molecular modifications affect the kinetics of 99m Tc-radiolabeled fatty acid compounds favorably. From the data set, rules for estimating the biodistribution of fatty acids tracers are deduced.     

Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.