Assembly, Characterization, and Delivery of Quantum Dot Labeled Biotinylated Lipid Particles

Lipid nanoparticles composed of mixtures of PEGylated-lipids; cationic and neutral lipids prepared by detergent dialysis can encapsulate biological active molecules and show considerable potential as systemic therapeutic agents. Addition of biotinylated lipids to this formulation allows surface modification of these particles with a suitable ligand or probe conjugated to streptavidin for specific cell targeting. Monitoring long circulating particles and cellular uptake requires stable and bright fluorescent probes. Quantum dots (QDs) constitute a relatively new class of fluorescent probes that overcome the limitations of organic fluorophores in biological imaging applications. Here, a protocol for the encapsulation of QD655 (red) in biotinylated lipid particles (BLPs) prepared by a detergent dialysis technique is presented followed by characterization of the loaded liposomal vehicles. Then, a protocol for BLPs surface modification via biotin-streptavidin linkage with preformed complexes of ligand-QD525 (green) for specific cell targeting of the nanoparticle is detailed. Conditions for cell binding and uptake of two colors QD labeled BLPs as well as basic microscopic settings for confocal live cell imaging are described

Notch-independent RBPJ controls angiogenesis in the adult heart

Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury

Subcellular homeostasis of phytohormone auxin is mediated by the ER-localized PIN5 transporter

The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues(1). Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell(2,3). In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients(2,4,5). Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum ( ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters ( including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants

Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm

Archaea are usually minor components of a microbial community and dominated by a large and diverse bacterial population. In contrast, the SM1 Euryarchaeon dominates a sulfidic aquifer by forming subsurface biofilms that contain a very minor bacterial fraction (5%). These unique biofilms are delivered in high biomass to the spring outflow that provides an outstanding window to the subsurface. Despite previous attempts to understand its natural role, the metabolic capacities of the SM1 Euryarchaeon remain mysterious to date. In this study, we focused on the minor bacterial fraction in order to obtain insights into the ecological function of the biofilm. We link phylogenetic diversity information with the spatial distribution of chemical and metabolic compounds by combining three different state-of-the-art methods: PhyloChip G3 DNA microarray technology, fluorescence in situ hybridization (FISH) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy. The results of PhyloChip and FISH technologies provide evidence for selective enrichment of sulfate-reducing bacteria, which was confirmed by the detection of bacterial dissimilatory sulfite reductase subunit B (dsrB) genes via quantitative PCR and sequence-based analyses. We further established a differentiation of archaeal and bacterial cells by SR-FTIR based on typical lipid and carbohydrate signatures, which demonstrated a co-localization of organic sulfate, carbonated mineral and bacterial signatures in the biofilm. All these results strongly indicate an involvement of the SM1 euryarchaeal biofilm in the global cycles of sulfur and carbon and support the hypothesis that sulfidic springs are important habitats for Earth's energy cycles. Moreover, these investigations of a bacterial minority in an Archaea-dominated environment are a remarkable example of the great power of combining highly sensitive microarrays with label-free infrared imaging