N-glycosylation is a potent regulator of prion protein neurotoxicity.

The C-terminal domain of the cellular prion protein (PrPC) contains two N-linked glycosylation sites, the occupancy of which impacts disease pathology. In this study, we demonstrate that glycans at these sites are required to maintain an intramolecular interaction with the N-terminal domain, mediated through a previously identified copper-histidine tether, which suppresses the neurotoxic activity of PrPC. NMR and electron paramagnetic resonance spectroscopy demonstrate that the glycans refine the structure of the protein's interdomain interaction. Using whole-cell patch-clamp electrophysiology, we further show that cultured cells expressing PrP molecules with mutated glycosylation sites display large, spontaneous inward currents, a correlate of PrP-induced neurotoxicity. Our findings establish a structural basis for the role of N-linked glycans in maintaining a nontoxic, physiological fold of PrPC.

High-resolution imaging of the osteogenic and angiogenic interface at the site of murine cranial bone defect repair via multiphoton microscopy.

The spatiotemporal blood vessel formation and specification at the osteogenic and angiogenic interface of murine cranial bone defect repair were examined utilizing a high-resolution multiphoton-based imaging platform in conjunction with advanced optical techniques that allow interrogation of the oxygen microenvironment and cellular energy metabolism in living animals. Our study demonstrates the dynamic changes of vessel types, that is, arterial, venous, and capillary vessel networks at the superior and dura periosteum of cranial bone defect, suggesting a differential coupling of the vessel type with osteoblast expansion and bone tissue deposition/remodeling during repair. Employing transgenic reporter mouse models that label distinct types of vessels at the site of repair, we further show that oxygen distributions in capillary vessels at the healing site are heterogeneous as well as time- and location-dependent. The endothelial cells coupling to osteoblasts prefer glycolysis and are less sensitive to microenvironmental oxygen changes than osteoblasts. In comparison, osteoblasts utilize relatively more OxPhos and potentially consume more oxygen at the site of repair. Taken together, our study highlights the dynamics and functional significance of blood vessel types at the site of defect repair, opening up opportunities for further delineating the oxygen and metabolic microenvironment at the interface of bone tissue regeneration.

Alzheimer's Drug PBT2 Interacts with the Amyloid ß 1-42 Peptide Differently than Other 8-Hydroxyquinoline Chelating Drugs.

Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid ß (Aß) peptide is central to disease pathology, which is supported by elevated Aß levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aß, and metal ion chelators have been shown to reverse this reaction in vitro. 8-Hydroxyquinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aß(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aß(1-42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aß(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aß(1-42) species in solution, leaving a single Cu(II)Aß(1-42) species. It follows that the Cu(II) site in this Cu(II)Aß(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aß(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aß(1-42).

NAD(P)H autofluorescence lifetime imaging enables single cell analyses of cellular metabolism of osteoblasts in vitro and in vivo via two-photon microscopy.

Two-photon fluorescence lifetime microscopy (2P-FLIM) is a non-invasive optical technique that can obtain cellular metabolism information based on the intrinsic autofluorescence lifetimes of free and enzyme-bound NAD(P)H, which reflect the metabolic state of single cells within the native microenvironment of the living tissue. NAD(P)H 2P-FLIM was initially performed in bone marrow stromal cell (BMSC) cultures established from Col (I) 2.3GFP or OSX-mCherry mouse models, in which osteoblastic lineage cells were labelled with green or red fluorescence protein, respectively. Measurement of the mean NAD(P)H lifetime, τM, demonstrated that osteoblasts in osteogenic media had a progressively increased τM compared to cells in regular media, suggesting that osteoblasts undergoing mineralization had higher NAD+/NAD(P)H ratio and may utilize more oxidative phosphorylation (OxPhos). In vivo NAD(P)H 2P-FLIM was conducted in conjunction with two-photon phosphorescence lifetime microscopy (2P-PLIM) to evaluate cellular metabolism of GFP+ osteoblasts as well as bone tissue oxygen at different locations of the native cranial bone in Col (I) 2.3GFP mice. Our data showed that osteocytes dwelling within lacunae had higher τM than osteoblasts at the bone edge of suture and marrow space. Measurement of pO2 showed poor correlation of pO2 and τM in native bone. However, when NAD(P)H 2P-FLIM was used to examine osteoblast cellular metabolism at the leading edge of the cranial defects during repair in Col (I) 2.3GFP mouse model, a significantly lower τM was recorded, which was associated with lower pO2 at an early stage of healing, indicating an impact of hypoxia on energy metabolism during bone tissue repair. Taken together, our current study demonstrates the feasibility of using non-invasive optical NAD(P)H 2P-FLIM technique to examine cellular energy metabolism at single cell resolution in living animals. Our data further support that both glycolysis and OxPhos are being used in the osteoblasts, with more mature osteoblasts exhibiting higher ratio of NAD+/NAD(P)H, indicating a potential change of energy mode during differentiation. Further experiments utilizing animals with genetic modification of cellular metabolism could enhance our understanding of energy metabolism in various cell types in living bone microenvironment.

Spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of biomimetic nanofiber-enabled bone tissue engineering.

While extensive research has demonstrated an interdependent role of osteogenesis and angiogenesis in bone tissue engineering, little is known about how functional blood vessel networks are organized to initiate and facilitate bone tissue regeneration. Building upon the success of a biomimetic composite nanofibrous construct capable of supporting donor progenitor cell-dependent regeneration, we examined the angiogenic response and spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of cranial bone defect repair utilizing high resolution multiphoton laser scanning microscopy (MPLSM) in conjunction with intravital imaging. We demonstrate here that the regenerative vasculature can be specified as arterial and venous capillary vessels based upon endothelial surface markers of CD31 and Endomucin (EMCN), with CD31+EMCN- vessels exhibiting higher flowrate and higher oxygen tension (pO2) than CD31+EMCN+ vessels. The donor osteoblast clusters are uniquely coupled to the sprouting CD31+EMCN+ vessels connecting to CD31+EMCN- vessels. Further analyses reveal differential vascular response and vessel type distribution in healing and non-healing defects, associated with changes of gene sets that control sprouting and morphogenesis of blood vessels. Collectively, our study highlights the key role of spatiotemporal vessel type distribution in bone tissue engineering, offering new insights for devising more effective vascularization strategies for bone tissue engineering.

Electrospun core-shell nanofibers with encapsulated enamel matrix derivative for guided periodontal tissue regeneration.

The osteogenic effect of a composite electrospun core-shell nanofiber membrane encapsulated with Emdogain® (EMD) was evaluated. The membrane was developed through coaxial electrospinning using polycaprolactone as the shell and polyethylene glycol as the core. The effects of the membrane on the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) were examined using Alizarin Red S staining and qRT-PCR. Characterization of the nanofiber membrane demonstrated core-shell morphology with a mean diameter of ~1 µm. Examination of the release of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) from core-shell nanofibers over a 22-day period showed improved release profile of encapsulated proteins as compared to solid nanofibers. When cultured on EMD-containing core-shell nanofibers, PDLSCs showed significantly improved osteogenic differentiation with increased Alizarin Red S staining and enhanced osteogenic gene expression, namely OCN, RUNX2, ALP, and OPN. Core-shell nanofiber membranes may improve outcomes in periodontal regenerative therapy through simultaneous mechanical barrier and controlled drug delivery function.

Both N-Terminal and C-Terminal Histidine Residues of the Prion Protein Are Essential for Copper Coordination and Neuroprotective Self-Regulation.

The cellular prion protein (PrPC) comprises two domains: a globular C-terminal domain and an unstructured N-terminal domain. Recently, copper has been observed to drive tertiary contact in PrPC, inducing a neuroprotective cis interaction that structurally links the protein's two domains. The location of this interaction on the C terminus overlaps with the sites of human pathogenic mutations and toxic antibody docking. Combined with recent evidence that the N terminus is a toxic effector regulated by the C terminus, there is an emerging consensus that this cis interaction serves a protective role, and that the disruption of this interaction by misfolded PrP oligomers may be a cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein's cis interaction, which helps to protect against neurotoxicity carried out by its N terminus. We show that simultaneous mutation of these histidines drastically weakens the cis interaction and enhances spontaneous cationic currents in cultured cells, the first C-terminal mutant to do so. Whereas previous studies suggested that Cu2+ coordination was localized solely to the protein's N-terminal domain, we find that both domains contribute equatorially coordinated histidine residue side-chains, resulting in a novel bridging interaction. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by sequestering copper from the C terminus. Our findings further establish a structural basis for PrPC's C-terminal regulation of its otherwise toxic N terminus.

Inhibition of the mitochondrial permeability transition improves bone fracture repair.

Bone fracture is accompanied by trauma, mechanical stresses, and inflammation - conditions known to induce the mitochondrial permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening leads to more inflammation, cell death and potentially to disruption of fracture repair. Here we performed a proof-of-concept study and tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. First, our in vitro experiments indicated pro-osteogenic and anti-inflammatory effects in osteoprogenitors upon CypD knock-out or pharmacological inhibition. Using a bone fracture model in mice, we observed that bone formation and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice or wild type mice treated with a CypD inhibitor, NIM811, when compared to controls. These effects were evident in young male but not female mice, however in older (13 month-old) female mice bone formation was also increased during fracture repair. In contrast to global CypD knock-out, mesenchymal lineage-specific (Prx1-Cre driven) CypD deletion did not result in improved fracture repair. Our findings implicate MPTP in bone fracture and suggest systemic CypD inhibition as a modality to promote fracture repair.

Production of Artificially Doubly Glycosylated, 15N Labeled Prion Protein for NMR Studies Using a pH-Scanning Volatile Buffer System.

Bacterially expressed proteins used in NMR studies lack glycans, and proteins from other organisms are neither 15N labeled nor glycosylated homogeneously. Here, we add two artificial glycans to uniformly 15N labeled prion protein using a buffer system that evolves over a pH range to accommodate the conflicting pH requirements of the substrate and enzymes without the need to fine-tune buffer conditions. NMR and CD spectroscopy of the protein indicates that the glycans do not influence its fold.

Electrospun Fiber Mesh for High-Resolution Measurements of Oxygen Tension in Cranial Bone Defect Repair.

Tissue oxygenation is one of the key determining factors in bone repair and bone tissue engineering. Adequate tissue oxygenation is essential for survival and differentiation of the bone-forming cells and ultimately the success of bone tissue regeneration. Two-photon phosphorescence lifetime microscopy (2PLM) has been successfully applied in the past to image oxygen distributions in tissue with high spatial resolution. However, delivery of phosphorescent probes into avascular compartments, such as those formed during early bone defect healing, poses significant problems. Here, we report a multifunctional oxygen-reporting fibrous matrix fabricated through encapsulation of a hydrophilic oxygen-sensitive, two-photon excitable phosphorescent probe, PtP-C343, in the core of fibers during coaxial electrospinning. The oxygen-sensitive fibers support bone marrow stromal cell growth and differentiation and at the same time enable real-time high-resolution probing of partial pressures of oxygen via 2PLM. The hydrophilicity of the probe facilitates its gradual release into the nearby microenvironment, allowing fibers to act as a vehicle for probe delivery into the healing tissue. In conjunction with a cranial defect window chamber model, which permits simultaneous imaging of the bone and neovasculature in vivo via two-photon laser scanning microscopy, the oxygen-reporting fibers provide a useful tool for minimally invasive, high-resolution, real-time 3D mapping of tissue oxygenation during bone defect healing, facilitating studies aimed at understanding the healing process and advancing design of tissue-engineered constructs for enhanced bone repair and regeneration.

Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally Relevant Mutations: Analysis by Cross-Linking, MS/MS, and NMR.

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.

X-ray Absorption Spectroscopy Investigations of Copper(II) Coordination in the Human Amyloid ß Peptide.

Alzheimer's disease (AD) is the main cause of age-related dementia and currently affects approximately 5.7 million Americans. Major brain changes associated with AD pathology include accumulation of amyloid beta (Aß) protein fragments and formation of extracellular amyloid plaques. Redox-active metals mediate oligomerization of Aß, and the resultant metal-bound oligomers have been implicated in the putative formation of harmful, reactive species that could contribute to observed oxidative damage. In isolated plaque cores, Cu(II) is bound to Aß via histidine residues. Despite numerous structural studies of Cu(II) binding to synthetic Aß in vitro, there is still uncertainty surrounding Cu(II) coordination in Aß. In this study, we used X-ray absorption spectroscopy (XAS) and high energy resolution fluorescence detected (HERFD) XAS to investigate Cu(II) coordination in Aß(1-42) under various solution conditions. We found that the average coordination environment in Cu(II)Aß(1-42) is sensitive to X-ray photoreduction, changes in buffer composition, peptide concentration, and solution pH. Fitting of the extended X-ray absorption fine structure (EXAFS) suggests Cu(II) is bound in a mixture of coordination environments in monomeric Aß(1-42) under all conditions studied. However, it was evident that on average only a single histidine residue coordinates Cu(II) in monomeric Aß(1-42) at pH 6.1, in addition to 3 other oxygen or nitrogen ligands. Cu(II) coordination in Aß(1-42) at pH 7.4 is similarly 4-coordinate with oxygen and nitrogen ligands, although an average of 2 histidine residues appear to coordinate at this pH. At pH 9.0, the average Cu(II) coordination environment in Aß(1-42) appears to be 5-coordinate with oxygen and nitrogen ligands, including two histidine residues.

Two-ply channels for faster wicking in paper-based microfluidic devices.

This article describes the development of porous two-ply channels for paper-based microfluidic devices that wick fluids significantly faster than conventional, porous, single-ply channels. The two-ply channels were made by stacking two single-ply channels on top of each other and were fabricated entirely out of paper, wax and toner using two commercially available printers, a convection oven and a thermal laminator. The wicking in paper-based channels was studied and modeled using a modified Lucas-Washburn equation to account for the effect of evaporation, and a paper-based titration device incorporating two-ply channels was demonstrated.

Paper and toner three-dimensional fluidic devices: programming fluid flow to improve point-of-care diagnostics.

We present a new method for fabricating three-dimensional paper-based fluidic devices that uses toner as a thermal adhesive to bond multiple layers of patterned paper together. The fabrication process is rapid, involves minimal equipment (a laser printer and a laminator) and produces complex channel networks with dimensions down to 1 mm. The devices can run multiple diagnostic assays on one or more samples simultaneously, can incorporate positive and negative controls and can be programmed to display the results of the assays in a variety of patterns. The patterns of the results can encode information, which could be used to identify counterfeit devices, identify samples, encrypt the results for patient privacy or monitor patient compliance.

Fully enclosed microfluidic paper-based analytical devices.

This article introduces fully enclosed microfluidic paper-based analytical devices (microPADs) fabricated by printing toner on the top and bottom of the devices using a laser printer. Enclosing paper-based microfluidic channels protects the channels from contamination, contains and protects reagents stored on the device, contains fluids within the channels so that microPADs can be handled and operated more easily, and reduces evaporation of solutions from the channels. These benefits extend the capabilities of microPADs for applications as low-cost point-of-care diagnostic devices.

Nitric oxide and protein nitration in the cystic fibrosis airway.

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.

Efficient reversed-phase purification of a hydrophobic reaction product following Mitsunobu-mediated glycosylation.

The Mitsunobu reaction was used to attach tetra-O-benzyl-D-glucopyranose to a monoindolylmaleimide, providing a key intermediate in the total synthesis of indolocarbazole topoisomerase I poisons. Using normal-phase silica gel chromatography, purification of the glycosylated product normally required multiple columns, resulting in poor recovered yields. Reversed-phase chromatography was used successfully to purify this highly hydrophobic material, rapidly and in high yield.