The cryoEM structure of the Hendra henipavirus nucleoprotein reveals insights into paramyxoviral nucleocapsid architectures.

We report the first cryoEM structure of the Hendra henipavirus nucleoprotein in complex with RNA, at 3.5 Å resolution, derived from single particle analysis of a double homotetradecameric RNA-bound N protein ring assembly exhibiting D14 symmetry. The structure of the HeV N protein adopts the common bi-lobed paramyxoviral N protein fold; the N-terminal and C-terminal globular domains are bisected by an RNA binding cleft containing six RNA nucleotides and are flanked by the N-terminal and C-terminal arms, respectively. In common with other paramyxoviral nucleocapsids, the lateral interface between adjacent Ni and Ni+1 protomers involves electrostatic and hydrophobic interactions mediated primarily through the N-terminal arm and globular domains with minor contribution from the C-terminal arm. However, the HeV N multimeric assembly uniquely identifies an additional protomer-protomer contact between the Ni+1 N-terminus and Ni-1 C-terminal arm linker. The model presented here broadens the understanding of RNA-bound paramyxoviral nucleocapsid architectures and provides a platform for further insight into the molecular biology of HeV, as well as the development of antiviral interventions.

Outer membrane utilisomes mediate glycan uptake in gut Bacteroidetes.

Bacteroidetes are abundant members of the human microbiota, utilizing a myriad of diet- and host-derived glycans in the distal gut1. Glycan uptake across the bacterial outer membrane of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, which is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan-binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the outer membrane are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilization systems of Bacteroides thetaiotaomicron, the additional outer membrane components assemble on the core SusCD transporter, forming stable glycan-utilizing machines that we term utilisomes. Single-particle cryogenic electron microscopy structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalize the role of each component in the utilisome.

Simple, Reliable Protocol for High-Yield Solubilization of Seedless Amyloid-ß Monomer.

Self-assembly of the amyloid-ß (Aß) peptide to form toxic oligomers and fibrils is a key causal event in the onset of Alzheimer's disease, and Aß is the focus of intense research in neuroscience, biophysics, and structural biology aimed at therapeutic development. Due to its rapid self-assembly and extreme sensitivity to aggregation conditions, preparation of seedless, reproducible Aß solutions is highly challenging, and there are serious ongoing issues with consistency in the literature. In this paper, we use a liquid-phase separation technique, asymmetric flow field-flow fractionation with multiangle light scattering (AF4-MALS), to develop and validate a simple, effective, economical method for re-solubilization and quality control of purified, lyophilized Aß samples. Our findings were obtained with recombinant peptide but are physicochemical in nature and thus highly relevant to synthetic peptide. We show that much of the variability in the literature stems from the inability of overly mild solvent treatments to produce consistently monomeric preparations and is rectified by a protocol involving high-pH (>12) dissolution, sonication, and rapid freezing to prevent modification. Aß treated in this manner is chemically stable, can be stored over long timescales at -80 °C, and exhibits remarkably consistent self-assembly behavior when returned to near-neutral pH. These preparations are highly monomeric, seedless, and do not require additional rounds of size exclusion, eliminating the need for this costly procedure and increasing the flexibility of use. We propose that our improved protocol is the simplest, fastest, and most effective way to solubilize Aß from diverse sources for sensitive self-assembly and toxicity assays.

Single Particle Cryo-Electron Microscopy: From Sample to Structure.

Cryo-electron microscopy (cryoEM) is a powerful technique for structure determination of macromolecular complexes, via single particle analysis (SPA). The overall process involves i) vitrifying the specimen in a thin film supported on a cryoEM grid; ii) screening the specimen to assess particle distribution and ice quality; iii) if the grid is suitable, collecting a single particle dataset for analysis; and iv) image processing to yield an EM density map. In this protocol, an overview for each of these steps is provided, with a focus on the variables which a user can modify during the workflow and the troubleshooting of common issues. With remote microscope operation becoming standard in many facilities, variations on imaging protocols to assist users in efficient operation and imaging when physical access to the microscope is limited will be described.

Insights into SusCD-mediated glycan import by a prominent gut symbiont.

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a "pedal bin" transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the ß2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism.

Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis.

Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a 'pedal bin' mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis.

Implantation Time Has No Effect on the Morphology and Extent of Previously Reported "Degradation" of Prolene Pelvic Mesh.

OBJECTIVES: Prolene polypropylene ("Prolene") meshes demonstrate no in vivo degradation, yet some claim degradation continues until no more Prolene polypropylene can be oxidized. We studied whether implantation time affects the morphology/extent of previously reported as cracking/degradation of completely cleaned Prolene explants. METHODS: Urogynecological explants (248 patients) were collected. After excluding non-Prolene/unknown meshes and those without known implantation times, completely cleaned explants (n = 205; 0.2-14.4 years implantation) were analyzed with light microscopy, scanning electron microscopy, and Fourier transform infrared spectroscopy. Based on implant times and storage (fixative or dry), representative specimens were randomly selected for comparison. Controls were unused ("exemplar") TVT specimens with and without intentional oxidation via ultraviolet light exposure. RESULTS: Prolene explants included 31 dry (18 TVT; 7 Prolift; 4 Gynemesh; 2 others) and 174 wet (87 TVT; 47 Prolift; 10 Gynemesh; 30 others) specimens. Specimens had similar morphologies before cleaning. Progressive cleaning removed tissue and cracked tissue-related material exposing smooth, unoxidized, and nondegraded fibers, with no visible gradient-type/ductile damage. Fourier transform infrared spectroscopy of the explants confirmed progressive loss of proteins. Cleaning intentionally oxidized exemplars did not remove oxidized carbonyl frequencies and showed deep cracks and gross fiber rupture/embrittlement, unlike the explants and nonoxidized exemplars. CONCLUSIONS: If in vivo Prolene degradation exists, there should be wide-ranging crack morphology and nonuniform crack penetration, as well as more cracking, degradation, and physical breakage for implants of longer implantation times, but this was not the case. There is no morphologic or spectral/chemical evidence of Prolene mesh degradation after up to 14.4 years in vivo.

Postmortem Retrieval Analysis of Metallosis and Periprosthetic Tissue Metal Concentrations in Total Knee Arthroplasty.

BACKGROUND: The purpose of this study is to determine the preferred sampling location for tissue analysis in total knee arthroplasty (TKA) and to evaluate metal concentrations, inflammatory cytokines, component damage, and tissue metallosis. METHODS: Twenty TKA systems were collected at necropsy along with tissue samples from 5 distinct locations. Inductively coupled plasma mass spectrometry (ICP-MS) analysis was performed to determine cobalt (Co), chromium (Cr), and titanium (Ti) concentrations. Synovial fluid cytokine analysis was preformed using a Magnetic Luminex Screening Assay. Femoral components were assesed for damage and tissues were visually scored for metallosis. RESULTS: The median metal concentrations were 16 ppb for Co, 46 ppb for Cr, and 9.8 ppb for Ti. There was no association between the tissue collection site and the metal concentration for Co (P = .979), Cr (P = .712), or Ti (P = .854). Twelve of 20 of the necropsy-retrieved TKAs had metallosis, but there was no correlation between Co (P = .48), Cr (P = .89), or Ti (P = .60) concentration and metallosis. Increased Co was associated with decreased tumor necrosis factor alpha (ρ = -0.56, P = .01) and interleukin 1 beta (ρ = -0.48, P = .03). Increased Cr was associated with decreased tumor necrosis factor alpha (ρ= -0.47, P = .03), interleukin 6 (ρ= -0.43, P = .04), and macrophage inflammatory protein 3 alpha (ρ= -0.47, P = .03). CONCLUSION: We observed elevated Co, Cr, and Ti concentrations in tissue from necropsy-retrieved TKA. Our findings did not support the hypothesis that tissue metal concentrations were associated with inflammatory cytokines. The results of this research will be useful for the design of future prospective studies.

The myth: in vivo degradation of polypropylene-based meshes.

INTRODUCTION AND HYPOTHESIS: Polypropylene is a base polymer used in biomaterial applications, including sutures and mesh products, for the treatment of pelvic organ prolapse, stress urinary incontinence, and hernia repairs. Previous studies have dismissed the value of formulation additives employed in polypropylene, and the importance and necessity of an effective mesh explant cleaning protocol when characterizing explanted devices. However, both are critical to understanding the alleged degradation of polypropylene-based meshes. METHODS: An effective, nondestructive, hydrolytic cleaning process, supplemented with light microscopy (LM), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) data, was used to evaluate 78 explanted Prolene meshes (with duration of implantation ranging from 0.4 to 11.7 years). RESULTS: The cleaning process exposed clean, unoxidized, nondegraded Prolene fibers with smooth surfaces and with no visible evidence of gradient-type or ductile damage. LM showed identical translucent and sometimes clear, cracked/flaking material on both blue and clear fibers, instead of clear cracked/flaking material on the clear fibers and blue cracked/flaking material on the blue fibers. FTIR confirmed progressive protein removal and loss of protein absorption intensity after each cleaning step. CONCLUSIONS: Our effective cleaning of explanted Prolene meshes and subsequent analyses showed that they did not degrade in vivo, confirming the in vivo stability of properly formulated polypropylene. Instead, the cracked layer that some researchers have identified as degraded Prolene is an adsorbed protein-formaldehyde coating, resulting from the well-established formalin-protein fixation process, which occurs immediately upon placing an explant in formalin.

Concomitant differentiation of a population of mouse embryonic stem cells into neuron-like cells and schwann cell-like cells in a slow-flow microfluidic device.

BACKGROUND: To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining spiral ganglion neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF-induced mouse embryonic stem cell (mESC)-derived "neurons" could potentially substitute for lost or damaged SGN. mESC-derived "Schwann cells" produce MIF, as do all Schwann cells (Huang et al., a; Roth et al., 2007; Roth et al., 2008) and could attract SGN to a "cell-coated" implant. RESULTS: Neuron- and Schwann cell-like cells were produced from a common population of mESCs in an ultra-slow-flow microfluidic device. As the populations interacted, "neurons" grew over the "Schwann cell" lawn, and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF-expressing "Schwann cells" were used to coat a CI: Mouse SGN and MIF-induced "neurons" grew directionally to the CI and to a wild-type but not MIF-knockout organ of Corti explant. CONCLUSIONS: Two novel stem cell-based approaches for treating the problem of sensorineural hearing loss are described. Developmental Dynamics 246:7-27, 2017. © 2016 Wiley Periodicals, Inc.

Aqueous two-phase system-mediated antibody micropatterning enables multiplexed immunostaining of cell monolayers and tissues.

Conventional immunostaining methods consume large quantities of expensive antibodies and are limited in terms of the number of antigens that can be detected from a single sample. In order to achieve multiplexed immunostaining, we micropatterned antibodies using aqueous two-phase systems (ATPS) formed from polyethylene glycol (PEG) and dextran. Multiple antigens can be detected on a single fixed sample by incorporating antibodies within dextran solutions, which are then patterned by micropipetting at specific sites on the sample in a solution of PEG. The antibodies are retained within the dextran phase due to biomolecular partitioning, allowing multiple protein markers to be visualized simultaneously by way of chromogenic, chemiluminescent, or immunofluorescent detection. This aqueous two-phase system-mediated antibody micropatterning approach allows antibody dilutions to be easily optimized, reduces the consumption of expensive primary antibodies and can prevent antibody cross-reactions, since the antibodies are retained at separate sites within the dextran microdroplets.

Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.

Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Cell co-culture patterning using aqueous two-phase systems.

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosis.

Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3'-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.

Receptor differential activation and cooperativity better explain cellular preference for different chemoattractant gradient shapes in an EGFR system.

This manuscript describes mathematical models that apply an aggregating receptor scheme to the epidermal growth factor receptor (EGFR) system to interpret and predict directed cell migration behaviors in differently-shaped chemoattractant gradients. This method incorporates the latest biochemical insights on ligand-receptor activation kinetics and receptor cooperativity into the commonly used difference in the fractional receptor occupancy (DFRO) model for explaining chemotaxis. The enhanced model derives the functionally more relevant value of difference in fractional receptor activation (DFRA). This DFRA analysis encompasses all features and predictions of the DFRO analyses. Importantly, DFRA analysis can additionally explain in vitro microfluidic chemotaxis experiments that are difficult to explain using only DFRO concepts such as why some cells may migrate well only in a higher concentration regime of exponential chemoattractant gradients. The DFRA analysis also suggests receptor activation strategies that cells may use to tune their responsiveness to differently-shaped in vivo gradients. DFRA analysis is conceptually and computationally straightforward. The results it provides are envisioned to serve as quick semi-quantitative guides to design chemotaxis experiments and to develop hypotheses for interpretation of results from directed cell migration experiments.

Responses of endothelial cells to extremely slow flows.

The process of blood vessel formation is accompanied by very minimal flow in the beginning, followed by increased flow rates once the vessel develops sufficiently. Many studies have been performed for endothelial cells at shear stress levels of 0.1-60 dyn∕cm(2); however, little is known about the effect of extremely slow flows (shear stress levels of 10(-4)-10(-2) dyn∕cm(2)) that endothelial cells may experience during early blood vessel formation where flow-sensing by indirect mass transport sensing rather than through mechanoreceptor sensing mechanisms would become more important. Here, we show that extremely low flows enhance proliferation, adherens junction protein localization, and nitric oxide secretion of endothelial cells, but do not induce actin filament reorganization. The responses of endothelial cells in different flow microenvironments need more attention because increasing evidence shows that endothelial cell behaviors at the extremely slow flow regimes cannot be linearly extrapolated from observations at faster flow rates. The devices and methods described here provide a useful platform for such studies.