Pseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool

Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making

Antigen-specific B-cell receptor sensitizes B cells to infection by influenza virus

Influenza A virus-specific B lymphocytes and the antibodies they produce protect against infection. However, the outcome of interactions between an influenza haemagglutinin-specific B cell via its receptor (BCR) and virus is unclear. Through somatic cell nuclear transfer we generated mice that harbour B cells with a BCR specific for the haemagglutinin of influenza A/WSN/33 virus (FluBI mice). Their B cells secrete an immunoglobulin gamma 2b that neutralizes infectious virus. Whereas B cells from FluBI and control mice bind equivalent amounts of virus through interaction of haemagglutinin with surface-disposed sialic acids, the A/WSN/33 virus infects only the haemagglutinin-specific B cells. Mere binding of virus is not sufficient for infection of B cells: this requires interactions of the BCR with haemagglutinin, causing both disruption of antibody secretion and FluBI B-cell death within 18 h. In mice infected with A/WSN/33, lung-resident FluBI B cells are infected by the virus, thus delaying the onset of protective antibody release into the lungs, whereas FluBI cells in the draining lymph node are not infected and proliferate. We propose that influenza targets and kills influenza-specific B cells in the lung, thus allowing the virus to gain purchase before the initiation of an effective adaptive response.National Institutes of Health (U.S.

Identification of host cell factors required for intoxication through use of modified cholera toxin

We describe a novel labeling strategy to site-specifically attach fluorophores, biotin, and proteins to the C terminus of the A1 subunit (CTA1) of cholera toxin (CTx) in an otherwise correctly assembled and active CTx complex. Using a biotinylated N-linked glycosylation reporter peptide attached to CTA1, we provide direct evidence that ∼12% of the internalized CTA1 pool reaches the ER. We also explored the sortase labeling method to attach the catalytic subunit of diphtheria toxin as a toxic warhead to CTA1, thus converting CTx into a cytolethal toxin. This new toxin conjugate enabled us to conduct a genetic screen in human cells, which identified ST3GAL5, SLC35A2, B3GALT4, UGCG, and ELF4 as genes essential for CTx intoxication. The first four encode proteins involved in the synthesis of gangliosides, which are known receptors for CTx. Identification and isolation of the ST3GAL5 and SLC35A2 mutant clonal cells uncover a previously unappreciated differential contribution of gangliosides to intoxication by CTx.Fundação para a Ciência e a Tecnologia (Fellowship

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong

Fungal recognition is mediated by the association of dectin-1 and galectin-3 in macrophages

Dectin-1, the major β-glucan receptor in leukocytes, triggers an effective immune response upon fungal recognition. Here we use sortase-mediated transpeptidation, a technique that allows placement of a variety of probes on a polypeptide backbone, to monitor the behavior of labeled functional dectin-1 in live cells with and without fungal challenge. Installation of probes on dectin-1 by sortagging permitted highly specific visualization of functional protein on the cell surface and its subsequent internalization upon ligand presentation. Retrieval of sortagged dectin-1 expressed in macrophages uncovered a unique interaction between dectin-1 and galectin-3 that functions in the proinflammatory response of macrophages to pathogenic fungi. When macrophages expressing dectin-1 are exposed to Candida albicans mutants with increased exposure of β-glucan, the loss of galectin-3 dramatically accentuates the failure to trigger an appropriate TNF-α response.National Institutes of Health (U.S.) (Grant GM040266)National Institute of Allergy and Infectious Diseases (U.S.) (Grant 5 R01 AI087879)Spain. Ministerio de Educación y Ciencia (Visiting Scholar Program (FU2006-0983))Fulbright ProgramNational Institutes of Health (U.S.) (National Research Service Award (F32 AI729353))Whitehead Institute for Biomedical Research (Margaret and Herman Sokol Fellowship in Biomedical Research

Preparation of unnatural N-to-N and C-to-C protein fusions

Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein–protein fusions, the method reported can be extended to connecting proteins with any entity of interest.Netherlands Organization for Scientific ResearchHuman Frontier Science Program (Strasbourg, France)National Institutes of Health (U.S.) (R01 Award AI033456)National Institutes of Health (U.S.) (R01 Award AI087879

Modified bibenzimidazole ligands as spectator ligands in photoactive molecular functional Ru-polypyridine units? Implications from spectroscopy

The photophysical properties of Ruthenium-bipyridine complexes bearing a bibenzimidazole ligand were investigated. The nitrogens on the bibenzimidazole-ligand were protected, by adding either a phenylene group or a 1,2-ethandiyl group, to remove the photophysical dependence of the complex on the protonation state of the bibenzimidazole ligand. This protection results in the bibenzimidazole ligand contributing to the MLCT transition, which is experimentally evidenced by (resonance) Raman scattering in concert with DFT calculations for a detailed mode assignment in the (resonance) Raman spectra

Pseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool

Background: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making. Methods: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue’s molecular fingerprint. Results: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology. Conclusions: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard