Physicochemical Analysis of Cold Brew and Hot Brew Peaberry Coffee

Peaberry coffee is the result of a natural mutation of coffee beans, and they make up only about 5–7% of coffee crops. A typical coffee cherry contains two seeds that are developed against each other, resulting in the distinctive half-rounded shape of coffee beans. However, failing to fertilize both ovules of one of the seeds or failure in endosperm development can cause only one of the seeds to develop, resulting in smaller, denser beans with a more domed shape. Peaberry coffees are said to be sweeter, lighter, and more flavorful since the peaberry beans receive all nutrients from the coffee cherry. Due to its exclusive nature, the chemical characteristic of peaberry coffee is not well understood. This study explores the acidities and antioxidant activity of peaberry coffee sourced from multiple regions. Total antioxidant capacity, total caffeoylquinic acid (CQA), total caffeine concentration, and pH levels were evaluated for peaberry coffee extracts prepared by cold and hot brewing methods. Little correlation between antioxidant activity and the concentrations of caffeine and CQA in peaberry beans was shown. Six methods were performed for the characterization of total antioxidant capacity including cyclic voltammetry, ABTS assay, and FRAP assay. Peaberry bean extract demonstrated higher average total caffeine concentrations compared to traditional coffee bean extracts

A Lipid Receptor Sorts Polyomavirus from the Endolysosome to the Endoplasmic Reticulum to Cause Infection

The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py) binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER) where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism

Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter

Discovery of Dual-Action Membrane-Anchored Modulators of Incretin Receptors

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function.Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G(7)]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G(7) into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G(7)]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes.These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target

The Anti-interferon Activity of Conserved Viral dUTPase ORF54 is Essential for an Effective MHV-68 Infection

Gammaherpesviruses such as KSHV and EBV establish lifelong persistent infections through latency in lymphocytes. These viruses have evolved several strategies to counteract the various components of the innate and adaptive immune systems. We conducted an unbiased screen using the genetically and biologically related virus, MHV-68, to find viral ORFs involved in the inhibition of type I interferon signaling and identified a conserved viral dUTPase, ORF54. Here we define the contribution of ORF54 in type I interferon inhibition by ectopic expression and through the use of genetically modified MHV-68. ORF54 and an ORF54 lacking dUTPase enzymatic activity efficiently inhibit type I interferon signaling by inducing the degradation of the type I interferon receptor protein IFNAR1. Subsequently, we show in vitro that the lack of ORF54 causes a reduction in lytic replication in the presence of type I interferon signaling. Investigation of the physiological consequence of IFNAR1 degradation and importance of ORF54 during MHV-68 in vivo infection demonstrates that ORF54 has an even greater impact on persistent infection than on lytic replication. MHV-68 lacking ORF54 expression is unable to efficiently establish latent infection in lymphocytes, although it replicates relatively normally in lung tissues. However, infection of IFNAR−/− mice alleviates this phenotype, emphasizing the specific role of ORF54 in type I interferon inhibition. Infection of mice and cells by a recombinant MHV-68 virus harboring a site specific mutation in ORF54 rendering the dUTPase inactive demonstrates that dUTPase enzymatic activity is not required for anti-interferon function of ORF54. Moreover, we find that dUTPase activity is dispensable at all stages of MHV-68 infection analyzed. Overall, our data suggest that ORF54 has evolved anti-interferon activity in addition to its dUTPase enzymatic activity, and that it is actually the anti-interferon role that renders ORF54 critical for establishing an effective persistent infection of MHV-68

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Klose R: Technical aspects of telepathology with emphasis on future development

Pathology undergoes presently changes due to new developments in diagnostic opportunities and cost saving efforts in health care. Out of the wide field of telepathology the paper selects three prototype applications: telepathology in teleeducation, expert advice for preselected details of a slide and finally telepathology for remote diagnosis. The most challenging field for remote diagnosis is the application in the frozen section scenario. The paper starts with the mental experiment to map conventional procedures to counterparts in telepathology. Technical opportunities and economical restrictions of telepathology equipment are discussed with respect to the components: electronic camera, display devices, haptic sensors and displays, available telecommunication channels and telepathology software. As an example and for illustration of the state of the art for an advanced telemicroscopy system able to perform remote frozen section diagnosis, the HISTKOM equipment is presented in more details. The section concerning future developments regards the aspects of the acceptance by tentative users, legal aspects, costs and affordability of equipment, the market for equipment components and the adequate telecommunication services. Further is regarded the mutual influence of properties of existing systems and application experiences gained with them on the next generation of equipment and application software. Conclusions and references close the paper

Technical Aspects of Telepathology with Emphasis on Future Development

Pathology undergoes presently changes due to new developments in diagnostic opportunities and cost saving efforts in health care. Out of the wide field of telepathology the paper selects three prototype applications: telepathology in teleeducation, expert advice for preselected details of a slide and finally telepathology for remote diagnosis. The most challenging field for remote diagnosis is the application in the frozen section scenario. The paper starts with the mental experiment to map conventional procedures to counterparts in telepathology