Domestic hot-water boilers harbour active thermophilic bacterial communities distinctly different from those in the cold-water supply.

Running cold and hot water in buildings is a widely established commodity. However, interests regarding hygiene and microbiological aspects had so far been focussed on cold water. Little attention has been given to the microbiology of domestic hot-water installations (DHWIs), except for aspects of pathogenic Legionella. World-wide, regulations consider hot (or warm) water as 'heated drinking water' that must comply (cold) drinking water (DW) standards. However, the few reports that exist indicate presence and growth of microbial flora in DHWIs, even when supplied with water with disinfectant residual. Using flow cytometric (FCM) total cell counting (TCC), FCM-fingerprinting, and 16S rRNA-gene-based metagenomic analysis, the characteristics and composition of bacterial communities in cold drinking water (DW) and hot water from associated boilers (operating at 50 - 60 °C) was studied in 14 selected inhouse DW installations located in Switzerland and Austria. A sampling strategy was applied that ensured access to the bulk water phase of both, supplied cold DW and produced hot boiler water. Generally, 1.3- to 8-fold enhanced TCCs were recorded in hot water compared to those in the supplied cold DW. FCM-fingerprints of cold and corresponding hot water from individual buildings indicated different composition of cold- and hot-water microbial floras. Also, hot waters from each of the boilers sampled had its own individual FCM-fingerprint. 16S rRNA-gene-based metagenomic analysis confirmed the marked differences in composition of microbiomes. E.g., in three neighbouring houses supplied from the same public network pipe each hot-water boiler contained its own thermophilic bacterial flora. Generally, bacterial diversity in cold DW was broad, that in hot water was restricted, with mostly thermophilic strains from the families Hydrogenophilaceae, Nitrosomonadaceae and Thermaceae dominating. Batch growth assays, consisting of cold DW heated up to 50 - 60 °C and inoculated with hot water, resulted in immediate cell growth with doubling times between 5 and 10 h. When cold DW was used as an inoculum no significant growth was observed. Even boilers supplied with UVC-treated cold DW contained an actively growing microbial flora, suggesting such hot-water systems as autonomously operating, thermophilic bioreactors. The generation of assimilable organic carbon from dissolved organic carbon due to heating appears to be the driver for growth of thermophilic microbial communities. Our report suggests that a man-made microbial ecosystem, very close to us all and of potential hygienic importance, may have been overlooked so far. Despite consumers having been exposed to microbial hot-water flora for a long time, with no major pathogens so far been associated specifically with hot-water usage (except for Legionella), the role of harmless thermophiles and their interaction with potential human pathogens able to grow at elevated temperatures in DHWIs remains to be investigated.

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

The Structure of the Mouse Serotonin 5-HT3 Receptor in Lipid Vesicles.

The function of membrane proteins is best understood if their structure in the lipid membrane is known. Here, we determined the structure of the mouse serotonin 5-HT3 receptor inserted in lipid bilayers to a resolution of 12 Å without stabilizing antibodies by cryo electron tomography and subtomogram averaging. The reconstruction reveals protein secondary structure elements in the transmembrane region, the extracellular pore, and the transmembrane channel pathway, showing an overall similarity to the available X-ray model of the truncated 5-HT3 receptor determined in the presence of a stabilizing nanobody. Structural analysis of the 5-HT3 receptor embedded in a lipid bilayer allowed the position of the membrane to be determined. Interactions between the densely packed receptors in lipids were visualized, revealing that the interactions were maintained by the short horizontal helices. In combination with methodological improvements, our approach enables the structural analysis of membrane proteins in response to voltage and ligand gating.

Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy.

We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use.

X-ray structure of the mouse serotonin 5-HT3 receptor.

Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.

Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy.

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellular communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells. Our purification strategy produced enriched samples of morphologically intact EVs free of extravesicular proteins and allowed labeling of marker molecules on the vesicle surface for single-vesicle analysis with single-molecule sensitivity. This novel approach provides information on the distribution profile of both EV size and relative expression level of a specific exosomal marker, deciphering the overall heterogeneity of EV preparations.

Downscaling the analysis of complex transmembrane signaling cascades to closed attoliter volumes.

Cellular signaling is classically investigated by measuring optical or electrical properties of single or populations of living cells. Here we show that ligand binding to cell surface receptors and subsequent activation of signaling cascades can be monitored in single, (sub-)micrometer sized native vesicles with single-molecule sensitivity. The vesicles are derived from live mammalian cells using chemicals or optical tweezers. They comprise parts of a cell's plasma membrane and cytosol and represent the smallest autonomous containers performing cellular signaling reactions thus functioning like minimized cells. Using fluorescence microscopies, we measured in individual vesicles the different steps of G-protein-coupled receptor mediated signaling like ligand binding to receptors, subsequent G-protein activation and finally arrestin translocation indicating receptor deactivation. Observing cellular signaling reactions in individual vesicles opens the door for downscaling bioanalysis of cellular functions to the attoliter range, multiplexing single cell analysis, and investigating receptor mediated signaling in multiarray format.

Protein-binding microarray analysis of tumor suppressor AP2α target gene specificity.

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.

Recombinant expression and functional characterization of mouse olfactory receptor mOR256-17 in mammalian cells.

Olfactory receptors (ORs) constitute the largest family of sensory membrane proteins in mammals. They play a key role within the olfactory system in recognizing and discriminating a nearly unlimited number of structurally diverse odorous molecules. The molecular basis of OR-mediated signal detection and transduction is poorly understood. This is due to difficulties in functional expression of ORs in high yields, preventing structural and biophysical studies at the level of the receptor protein. Here we report on recombinant expression of mouse receptor mOR256-17 yielding 10(6) ORs per cell in transiently transfected mammalian cells. For quantification and optimization of OR expression, we employed different fluorescent probes. Green fluorescent protein fused to the C-terminus of mOR256-17 allowed quantification of total cellular OR biosynthesis, and post-translational fluorescence labeling of a 12-amino acid polypeptide sequence at the N-terminus permitted the selective visualization and quantification of ORs at the plasma membrane using cell flow cytometry. Our dual-color labeling approach is generally applicable to quantification of membrane proteins for mammalian cell-based expression. By screening a large odorant compound library, we discovered a selective spectrum of potent mOR256-17-specific agonists essential for probing the receptor function for future scaled-up productions.

Deamidation and transamidation of substance P by tissue transglutaminase revealed by electron-capture dissociation fourier transform mass spectrometry.

Tissue transglutaminase (tTGase) catalyzes both deamidation and transamidation of peptides and proteins by using a peptidyl glutamine as primary substrate. A precise consensus sequence for the enzyme is unknown and the ratio between deamidated and transamidated (or cross-linked) reaction products is highly substrate-dependent. Due to its overlapping body distribution with tTGase and ease of manipulation with tandem mass spectrometry, we used the neuropeptide substance P as a model to investigate the associated enzymatic kinetics and reaction products. Online liquid-chromatography Fourier-transform ion-cyclotron-resonance mass spectrometry (FT-ICR MS) combined with electron-capture dissociation (ECD) was employed to study the tTGase-induced modifications of substance P. A particular strength of ECD for peptide-enzyme reaction product monitoring is its ability to distinguish isomeric amino acids, for example, Glu and iso-Glu, by signature product ions. Our studies show that the primary reaction observed is deamidation, with the two consecutive glutamine residues converted sequentially into glutamate: first Gln(5) , and subsequently Gln(6) . We then applied ECD FT-ICR MS to identify the transamidation site on an enzymatically cross-linked peptide, which turned out to correspond to Gln(5) . Three populations of substance-P dimers were detected that differed by the number of deamidated Gln residues. The higher reactivity of Gln(5) over Gln(6) was further confirmed by cross-linking SP with monodansylcadaverine (MDC). Overall, our approach described herein is of a general importance for mapping both enzymatically induced post-translational protein modifications and cross-linking. Finally, in vitro Ca-signaling assays revealed that the main tTGase reaction product, the singly deamidated SP (RPKPEQFFGLM-NH(2) ), has increased agonist potency towards its natural receptor, thus confirming the biologically relevant role of deamidation.