Multiplexed immuno-precipitation with 1725 commercially available antibodies to cellular proteins

Antibody array analysis of complex samples requires capture reagents with exceptional specificity. The frequency of antibodies with label-based detection may be as low as 5%. Here, however, we show that as many as 25% of commercially available antibodies are useful when biotinylated cellular proteins are fractionated by size exclusion chromatography (SEC) first. A microsphere multiplex with 1725 antibodies to cellular proteins was added to 24 SEC fractions, labelled with streptavidin and analyzed by flow cytometry (microsphere-based affinity proteomics, MAP) The SEC-MAP approach resolved different targets captured by each antibody as reactivity peaks across the separation range of the SEC column (10-670kDa). Complex reactivity profiles demonstrated that most antibodies bound more than one target. However, specific binding was readily detected as reactivity peaks common for different antibodies to the same protein. We optimized sample preparation and found that amine-reactive biotin rarely inhibited antibody binding when the biotin to lysine ratio was kept below 1:1 during labelling. Moreover, several epitopes that were inaccessible to antibodies in native proteins were unmasked after heat denaturation with 0.1% of SDS. The SEC-MAP format should allow researchers to build multiplexed assays with antibodies purchased for use in e.g. Western blotting.     

Lack of the receptor for advanced glycation end-products attenuates E. coli pneumonia in mice

Background

The receptor for advanced glycation end-products (RAGE) has been suggested to modulate lung injury in models of acute pulmonary inflammation. To study this further, model systems utilizing wild type and RAGE knockout (KO) mice were used to determine the role of RAGE signaling in lipopolysaccharide (LPS) and E. coli induced acute pulmonary inflammation. The effect of intraperitoneal (i.p.) and intratracheal (i.t.) administration of mouse soluble RAGE on E. coli injury was also investigated.

Methodology/Principal Findings

C57BL/6 wild type and RAGE KO mice received an i.t. instillation of LPS, E. coli, or vehicle control. Some groups also received i.p. or i.t. administration of mouse soluble RAGE. After 24 hours, the role of RAGE expression on inflammation was assessed by comparing responses in wild type and RAGE KO. RAGE protein levels decreased in wild type lung homogenates after treatment with either LPS or bacteria. In addition, soluble RAGE and HMGB1 increased in the BALF after E. coli instillation. RAGE KO mice challenged with LPS had the same degree of inflammation as wild type mice. However, when challenged with E. coli, RAGE KO mice had significantly less inflammation when compared to wild type mice. Most cytokine levels were lower in the BALF of RAGE KO mice compared to wild type mice after E. coli injury, while only monocyte chemotactic protein-1, MCP-1, was lower after LPS challenge. Neither i.p. nor i.t. administration of mouse soluble RAGE attenuated the severity of E. coli injury in wild type mice.

Conclusions/Significance

Lack of RAGE in the lung does not protect against LPS induced acute pulmonary inflammation, but attenuates injury following live E. coli challenge. These findings suggest that RAGE mediates responses to E. coli-associated pathogen-associated molecular pattern molecules other than LPS or other bacterial specific signaling responses. Soluble RAGE treatment had no effect on inflammation.     

Real-time ichthyoplankton drift in Northeast Arctic cod and Norwegian spring-spawning herring

Background

Individual-based biophysical larval models, initialized and parameterized by observations, enable numerical investigations of various factors regulating survival of young fish until they recruit into the adult population. Exponentially decreasing numbers in Northeast Arctic cod and Norwegian Spring Spawning herring early changes emphasizes the importance of early life history, when ichthyoplankton exhibit pelagic free drift. However, while most studies are concerned with past recruitment variability it is also important to establish real-time predictions of ichthyoplankton distributions due to the increasing human activity in fish habitats and the need for distribution predictions that could potentially improve field coverage of ichthyoplankton.

Methodology/Principal Findings

A system has been developed for operational simulation of ichthyoplankton distributions. We have coupled a two-day ocean forecasts from the Norwegian Meteorological Institute with an individual-based ichthyoplankton model for Northeast Arctic cod and Norwegian Spring Spawning herring producing daily updated maps of ichthyoplankton distributions. Recent years observed spawning distribution and intensity have been used as input to the model system. The system has been running in an operational mode since 2008. Surveys are expensive and distributions of early stages are therefore only covered once or twice a year. Comparison between model and observations are therefore limited in time. However, the observed and simulated distributions of juvenile fish tend to agree well during early fall. Area-overlap between modeled and observed juveniles September 1^st range from 61 to 73%, and 61 to 71% when weighted by concentrations.

Conclusions/Significance

The model system may be used to evaluate the design of ongoing surveys, to quantify the overlap with harmful substances in the ocean after accidental spills, as well as management planning of particular risky operations at sea. The modeled distributions are already utilized during research surveys to estimate coverage success of sampled biota and immediately after spills from ships at sea.     

Brain signal predictions from multi-scale networks using a linearized framework

Simulations of neural activity at different levels of detail are ubiquitous in modern neurosciences, aiding the interpretation of experimental data and underlying neural mechanisms at the level of cells and circuits. Extracellular measurements of brain signals reflecting transmembrane currents throughout the neural tissue remain commonplace. The lower frequencies (≲ 300Hz) of measured signals generally stem from synaptic activity driven by recurrent interactions among neural populations and computational models should also incorporate accurate predictions of such signals. Due to limited computational resources, large-scale neuronal network models (≳ 10⁶ neurons or so) often require reducing the level of biophysical detail and account mainly for times of action potentials (‘spikes’) or spike rates. Corresponding extracellular signal predictions have thus poorly accounted for their biophysical origin.Here we propose a computational framework for predicting spatiotemporal filter kernels for such extracellular signals stemming from synaptic activity, accounting for the biophysics of neurons, populations, and recurrent connections. Signals are obtained by convolving population spike rates by appropriate kernels for each connection pathway and summing the contributions. Our main results are that kernels derived via linearized synapse and membrane dynamics, distributions of cells, conduction delay, and volume conductor model allow for accurately capturing the spatiotemporal dynamics of ground truth extracellular signals from conductance-based multicompartment neuron networks. One particular observation is that changes in the effective membrane time constants caused by persistent synapse activation must be accounted for.The work also constitutes a major advance in computational efficiency of accurate, biophysics-based signal predictions from large-scale spike and rate-based neuron network models drastically reducing signal prediction times compared to biophysically detailed network models. This work also provides insight into how experimentally recorded low-frequency extracellular signals of neuronal activity may be approximately linearly dependent on spiking activity. A new software tool LFPykernels serves as a reference implementation of the framework.     

The plastid division protein AtMinD1 is a Ca2+-ATPase stimulated by AtMinE1

Bacteria and plastids divide symmetrically through binary fission by accurately placing the division site at midpoint, a process initiated by FtsZ polymerization, which forms a Z-ring. In Escherichia coli precise Z-ring placement at midcell depends on controlled oscillatory behavior of MinD and MinE: In the presence of ATP MinD interacts with the FtsZ inhibitor MinC and migrates to the membrane where the MinD-MinC complex recruits MinE, followed by MinD-mediated ATP hydrolysis and membrane release. Although correct Z-ring placement during Arabidopsis plastid division depends on the precise localization of the bacterial homologs AtMinD1 and AtMinE1, the underlying mechanism of this process remains unknown. Here we have shown that AtMinD1 is a Ca2+-dependent ATPase and through mutation analysis demonstrated the physiological importance of this activity where loss of ATP hydrolysis results in protein mislocalization within plastids. The observed mislocalization is not due to disrupted AtMinD1 dimerization, however; the active site AtMinD1(K72A) mutant is unable to interact with the topological specificity factor AtMinE1. We have shown that AtMinE1, but not E. coli MinE, stimulates AtMinD1-mediated ATP hydrolysis, but in contrast to prokaryotes stimulation occurs in the absence of membrane lipids. Although AtMinD1 appears highly evolutionarily conserved, we found that important biochemical and cell biological properties have diverged. We propose that correct intraplastidic AtMinD1 localization is dependent on AtMinE1-stimulated, Ca2+-dependent AtMinD1 ATP hydrolysis, ultimately ensuring precise Z-ring placement and symmetric plastid division.     

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

A homolog of Albino3/OxaI is essential for thylakoid biogenesis in the cyanobacterium Synechocystis sp. PCC6803

YidC/OxaI play essential roles in the insertion of a wide range of membrane proteins in Eschericha coli and mitochondria, respectively. In contrast, the chloroplast thylakoid homolog Albino3 (Alb3) facilitates the insertion of only a specialized subset of proteins, and the vast majority insert into thylakoids by a pathway that is so far unique to chloroplasts. In this study, we have analyzed the role of Alb3 in the cyanobacterium Synechocystis sp. PCC6803, which contains internal thylakoids that are similar in some respects to those of chloroplasts. The single alb3 gene (slr1471) was disrupted by the introduction of an antibiotic cassette, and photoautotrophic growth resulted in the generation of a merodiploid species (but not full segregation), indicating an essential role for Alb3 in maintaining the photosynthetic apparatus. Thylakoid organization is lost under these conditions, and the levels of photosynthetic pigments fall to approximately 40% of wild-type levels. Photosynthetic electron transport and oxygen evolution are reduced by a similar extent. Growth on glucose relieves the selective pressure to maintain photosynthetic competence, and under these conditions, the cells become completely bleached, again indicating that Alb3 is essential for thylakoid biogenesis. Full segregation could not be achieved under any growth regime, strongly suggesting that the slr1471 open reading frame is essential for cell viability.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

Operational concept for the improved synthesis of (R)-3,3'-furoin and related hydrophobic compounds with benzaldehyde lyase

Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.