Characterization of a Peptide that Specifically Blocks the Ras Binding Domain of p75

The neurotrophin receptor p75 interacts with the GTPase Ras. Unstimulated it inactivates Ras while ligand binding induces Ras activation. We developed an inhibitory peptide (ip75RBD) which interferes with the binding domain of Ras of the intracellular domain of p75. ip75RBD inhibits the binding of Ras to the receptor in vitro. It is membrane-permeable and inhibits ligand-induced Ras activation via p75 in vivo but does not influence Ras activation by the stimulated receptor tyrosine kinases Trk and the epidermal growth factor receptor EGFR. The activation of the neutral sphingomyelinase by stimulated p75 is slightly delayed but not inhibited by the peptide. p75-mediated neuronal death induced by NGF or aggregated beta-amyloid_1–42 is reduced. We conclude that ip75RBD specifically blocks the Ras binding site of p75 and can be used to analyze p75-induced Ras signaling.     

Enzymatic coating of lignocellulosic surfaces with polyphenols

Upgrading of the surface characteristics could enhance the bulk properties of naturally abundant fiber-forming materials for better performance or create new value-added products. Laccase can induce cross-linkage and covalent coupling of low molecular weight compounds onto lignocellulosic surfaces. For this purpose the 38-kDa laccase from Trametes hirsuta was purified and characterized. The best conditions for laccase-induced coating of flax fibers were determined. This evaluation was based on the obtained coloration and color depth. A screening was carried out with different phenols for their potential as monomers for enzyme-catalyzed polymerization resulting in a coating with antibacterial performance. While all the methoxyphenols showed different coloration with weak fastness properties, bacterial growth of Bacillus subtilis and Staphylococcus aureus was reduced significantly using ferulic acid and hydroquinone. Using laccase-induced coupling and polymerization, multi-functionality of the lignocellulosic surface, such as coloration and antimicrobial performance, was achieved, which depended on the nature of the applied phenolic monomer.     

TreeQ-VISTA: an interactive tree visualization tool with functional annotation query capabilities

We describe a general multiplatform exploratory tool called TreeQ-Vista, designed for presenting functional annotations in a phylogenetic context. Traits, such as phenotypic and genomic properties, are interactively queried from a user-provided relational database with a user-friendly interface which provides a set of tools for users with or without SQL knowledge. The query results are projected onto a phylogenetic tree and can be displayed in multiple color groups. A rich set of browsing, grouping and query tools are provided to facilitate trait exploration, comparison and analysis. AVAILABILITY: The program, detailed tutorial and examples are available online (http:/genome.lbl.gov/vista/TreeQVista).     

Sterol, protein and lipid trafficking in Chinese hamster ovary cells with Niemann-Pick type C1 defect

We studied the trafficking of sterols, lipids and proteins in Niemann-Pick type C (NPC) cells. The NPC is an inherited disorder involving the accumulation of sterol and lipids in modified late-endosome/lysosome-like storage organelles. Most sterol accumulation studies in NPC cells have been carried out using low-density lipoprotein (LDL) as the sterol source, and it has been shown that sterol efflux from late endosomes is impaired in NPC cells. In this study, we used a fluorescent sterol analog, dehydroergosterol, which can be quickly and efficiently delivered to the plasma membrane. Thus, we were able to study the trafficking kinetics of the non-LDL-derived sterol pool, and we found that dehydroergosterol accumulates in the storage organelles over the course of several hours in NPC cells. We also found that dialkylindocarbocyanine lipid-mimetic analogs that recycle efficiently from early endosomes in wild-type cells are targeted to late endosomal organelles in NPC cells, and transferrin receptors recycle slowly and inefficiently in NPC cells. These data are consistent with multiple trafficking defects in both early and late endosomes in NPC cells.     

Group Selection and Contribution of Minority Variants during Virus Adaptation Determines Virus Fitness and Phenotype

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

Rewiring of sIgM-Mediated Intracellular Signaling through the CD180 Toll-like Receptor

Chronic lymphocytic leukemia (CLL) development and progression are thought to be driven by unknown antigens/autoantigens through the B cell receptor (BCR) and environmental signals for survival and expansion including toll-like receptor (TLR) ligands. CD180/RP105, a membrane-associated orphan receptor of the TLR family, induces normal B cell activation and proliferation and is expressed by approximately 60% of CLL samples. Half of these respond to ligation with anti-CD180 antibody by increased activation/phosphorylation of protein kinases associated with BCR signaling. Hence CLL cells expressing both CD180 and the BCR could receive signals via both receptors. Here we investigated cross-talk between BCR and CD180-mediated signaling on CLL cell survival and apoptosis. Our data indicate that ligation of CD180 on responsive CLL cells leads to activation of either prosurvival Bruton tyrosine kinase (BTK)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT-mediated, or proapoptotic p38 mitogen-activated protein kinase (p38MAPK)-mediated signaling pathways, while selective immunoglobulin M (sIgM) ligation predominantly engages the BTK/PI3K/AKT pathway. Furthermore, pretreatment of CLL cells with anti-CD180 redirects IgM-mediated signaling from the prosurvival BTK/PI3K/AKT toward the proapoptotic p38MAPK pathway. Thus preengaging CD180 could prevent further prosurvival signaling mediated via the BCR and, instead, induce CLL cell apoptosis, opening the door to therapeutic profiling and new strategies for the treatment of a substantial cohort of CLL patients.     

Effects of climate extremes on the terrestrial carbon cycle: concepts,processes and potential future impacts

Extreme droughts, heat waves, frosts, precipitation, wind storms and other climate extremes may impact the structure, composition and functioning of terrestrial ecosystems, and thus carbon cycling and its feedbacks to the climate system. Yet, the interconnected avenues through which climate extremes drive ecological and physiological processes and alter the carbon balance are poorly understood. Here, we review the literature on carbon cycle relevant responses of ecosystems to extreme climatic events. Given that impacts of climate extremes are considered disturbances, we assume the respective general disturbance‐induced mechanisms and processes to also operate in an extreme context. The paucity of well‐defined studies currently renders a quantitative meta‐analysis impossible, but permits us to develop a deductive framework for identifying the main mechanisms (and coupling thereof) through which climate extremes may act on the carbon cycle. We find that ecosystem responses can exceed the duration of the climate impacts via lagged effects on the carbon cycle. The expected regional impacts of future climate extremes will depend on changes in the probability and severity of their occurrence, on the compound effects and timing of different climate extremes, and on the vulnerability of each land‐cover type modulated by management. Although processes and sensitivities differ among biomes, based on expert opinion, we expect forests to exhibit the largest net effect of extremes due to their large carbon pools and fluxes, potentially large indirect and lagged impacts, and long recovery time to regain previous stocks. At the global scale, we presume that droughts have the strongest and most widespread effects on terrestrial carbon cycling. Comparing impacts of climate extremes identified via remote sensing vs. ground‐based observational case studies reveals that many regions in the (sub‐)tropics are understudied. Hence, regional investigations are needed to allow a global upscaling of the impacts of climate extremes on global carbon–climate feedbacks.     

Ultrafast Charge Generation Pathways in Photovoltaic Blends Based on Novel Star‐Shaped Conjugated Molecules

The quest for new materials is one of the main factors propelling recent advances in organic photovoltaics. Star‐shaped small molecules (SSMs) have been proven promising candidates as perspective donor material due to the increase in numbers of excitation pathways caused by the degeneracy of the lowest unoccupied molecular orbital (LUMO) level. In order to unravel the pathways of the initial photon‐to‐charge conversion, the photovoltaic blends based on three different SSMs with a generic structure of N(phenylene‐nthiophene‐dicyanovinyl‐alkyl)₃ (n = 1–3), and [6,6]‐phenyl‐C₇₁‐butyric acid methyl ester (PC₇₁BM) acceptor are investigated by ultrafast photoinduced absorption spectroscopy assisted by density functional theory calculations. It is shown that both electron transfer from SSMs to PC₇₁BM and hole transfer from PC₇₁BM to SSMs are equally significant for generation of long‐lived charges. In contrast, intramolecular (intra‐SSM) charge separation results in geminate recombination and therefore constitutes a loss channel. Overall, up to 60% of long‐lived separated charges are generated at the optimal PC₇₁BM concentrations. The obtained results suggest that further improvement of the SSM‐based solar cells is feasible via optimization of blend morphology and by suppressing the intra‐SSM recombination channel.