Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p)

The insula cortex contacts distinct output streams of the central amygdala

The emergence of genetic tools has provided new means of mapping functionality in central amygdala (CeA) neuron populations based on their molecular profiles, response properties, and importantly, connectivity patterns. While abundant evidence indicates that neuronal signals arrive in the CeA eliciting both aversive and appetitive behaviors, our understanding of the anatomy of the underlying long-range CeA network remains fragmentary. In this study, we combine viral tracings, electrophysiological, and optogenetic approaches to establish in male mice, a wiring chart between the insula cortex (IC), a major sensory input region of the lateral and capsular part of the CeA (CeL/C), and four principal output streams of this nucleus. We found that retrogradely labeled output neurons occupy discrete and likely strategic locations in the CeL/C, and that they are disproportionally controlled by the IC. We identified a direct line of connection between the IC and the lateral hypothalamus (LH), which engages numerous LH-projecting CeL/C cells whose activity can be strongly upregulated on firing of IC neurons. In comparison, CeL/C neurons projecting to the bed nucleus of the stria terminalis (BNST) are also frequently contacted by incoming IC axons, but the strength of this connection is weak. Our results provide a link between long-range inputs and outputs of the CeA and pave the way to a better understanding of how internal, external, and experience dependent information may impinge on action selection by the CeA

The Process-Interaction-Model: a common representation of rule-based and logical models allows studying signal transduction on different levels of detail

BACKGROUND: Signaling systems typically involve large, structured molecules each consisting of a large number of subunits called molecule domains. In modeling such systems these domains can be considered as the main players. In order to handle the resulting combinatorial complexity, rule-based modeling has been established as the tool of choice. In contrast to the detailed quantitative rule-based modeling, qualitative modeling approaches like logical modeling rely solely on the network structure and are particularly useful for analyzing structural and functional properties of signaling systems. RESULTS: We introduce the Process-Interaction-Model (PIM) concept. It defines a common representation (or basis) of rule-based models and site-specific logical models, and, furthermore, includes methods to derive models of both types from a given PIM. A PIM is based on directed graphs with nodes representing processes like post-translational modifications or binding processes and edges representing the interactions among processes. The applicability of the concept has been demonstrated by applying it to a model describing EGF insulin crosstalk. A prototypic implementation of the PIM concept has been integrated in the modeling software ProMoT. CONCLUSIONS: The PIM concept provides a common basis for two modeling formalisms tailored to the study of signaling systems: a quantitative (rule-based) and a qualitative (logical) modeling formalism. Every PIM is a compact specification of a rule-based model and facilitates the systematic set-up of a rule-based model, while at the same time facilitating the automatic generation of a site-specific logical model. Consequently, modifications can be made on the underlying basis and then be propagated into the different model specifications – ensuring consistency of all models, regardless of the modeling formalism. This facilitates the analysis of a system on different levels of detail as it guarantees the application of established simulation and analysis methods to consistent descriptions (rule-based and logical) of a particular signaling system

Real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

Two real-time PCR assays for detection of Mycolasma hyopneumoniae (Mhyop) in clinical lung samples were established and validated in parallel. One is targeting a repetitive DNA element (REP assay) the other a putative ABC transporter gene (ABC assay). The two assays were shown to be 100% specific when testing pig lungs from defined negative farms. When investigating defined positive farms the REP assay tested with a sensitivity of about 50%, the ABC assay with 90%. The two assays together, however detected 100% of positive farms. Within a single positive farm on average 90% of the samples tested positive with the REP or ABC assay. Analysing a set of 41 lungs from infected pigs from routine diagnostic the REP assay detected 50% and the ABC assay 70%, while both assays together had a sensitivity of 85%

Immunogenicity Studies in Carnivores Using a Rabies Virus Construct with a Site-Directed Deletion in the Phosphoprotein

Different approaches have been applied to develop highly attenuated rabies virus vaccines for oral vaccination of mesocarnivores. One prototype vaccine construct is SAD dIND1, which contains a deletion in the P-gene severely limiting the inhibition of type-1 interferon induction. Immunogenicity studies in foxes and skunks were undertaken to investigate whether this highly attenuated vaccine would be more immunogenic than the parental SAD B19 vaccine strain. In foxes, it was demonstrated that SAD dIND1 protected the animals against a rabies infection after a single oral dose, although virus neutralizing antibody titres were lower than in foxes orally vaccinated with the SAD B19 virus as observed in previous experiments. In contrast, skunks receiving 107.5 FFU SAD dIND1 did not develop virus neutralizing antibodies and were not protected against a subsequent rabies infection

A serial multiplex immunogold labeling method for identifying peptidergic neurons in connectomes.

This is the final version of the article.Available from eLife Sciences Publications via the DOI in this record.Electron microscopy-based connectomics aims to comprehensively map synaptic connections in neural tissue. However, current approaches are limited in their capacity to directly assign molecular identities to neurons. Here, we use serial multiplex immunogold labeling (siGOLD) and serial-section transmission electron microscopy (ssTEM) to identify multiple peptidergic neurons in a connectome. The high immunogenicity of neuropeptides and their broad distribution along axons, allowed us to identify distinct neurons by immunolabeling small subsets of sections within larger series. We demonstrate the scalability of siGOLD by using 11 neuropeptide antibodies on a full-body larval ssTEM dataset of the annelid Platynereis. We also reconstruct a peptidergic circuitry comprising the sensory nuchal organs, found by siGOLD to express pigment-dispersing factor, a circadian neuropeptide. Our approach enables the direct overlaying of chemical neuromodulatory maps onto synaptic connectomic maps in the study of nervous systems.The research leading to these results received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821. This project is supported by the Marie Curie ITN "Neptune", GA 317172, funded under the FP7, PEOPLE Work Programme of the European Commission

Neuronal connectome of a sensory-motor circuit for visual navigation.

This is the final version of the article. Available from eLife Sciences Publications via the DOI in this record.Animals use spatial differences in environmental light levels for visual navigation; however, how light inputs are translated into coordinated motor outputs remains poorly understood. Here we reconstruct the neuronal connectome of a four-eye visual circuit in the larva of the annelid Platynereis using serial-section transmission electron microscopy. In this 71-neuron circuit, photoreceptors connect via three layers of interneurons to motorneurons, which innervate trunk muscles. By combining eye ablations with behavioral experiments, we show that the circuit compares light on either side of the body and stimulates body bending upon left-right light imbalance during visual phototaxis. We also identified an interneuron motif that enhances sensitivity to different light intensity contrasts. The Platynereis eye circuit has the hallmarks of a visual system, including spatial light detection and contrast modulation, illustrating how image-forming eyes may have evolved via intermediate stages contrasting only a light and a dark field during a simple visual task.The research leading to these results received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/European Research Council Grant Agreement 260821

Poland Becoming a Member of the Global Nuclear Energy Partnership, Vol. 2.

Within a constrained carbon environment, the risks of future natural gas supply, and the need to move to market-based electricity prices, the study team found: (1) the deployment of new nuclear energy in Poland itself is very competitive in the next decade or two; (2) if such generation could be made available to Poland prior to deployment of its own nuclear generation facilities, Poland would benefit from partnering with its Baltic neighbors to import electricity derived from new nuclear generation facilities sited in Lithuania; and (3) Poland appears to be a good candidate for a partnership in the Global Nuclear Energy Partnership (GNEP) as an emerging nuclear energy country