Stable Isotope Labelling Kinetics (SILK) tracing of neurofilament light in human cerebrospinal fluid and brain tissue.

Background: Neurofilament light protein (NfL) is a promising biomarker of neuronal injury and neurodegeneration. NfL levels in cerebrospinal fluid (CSF) and blood provide information about disease progression and are increasingly relied on as outcome measure in clinical trials. Understanding NfL kinetics is critical for interpreting NfL in response to new events where a steady state cannot be assumed, such as acute injury, disease onset or progression, or response to disease‐modifying therapies.

Importance of CSF-based Aβ clearance with age in humans increases with declining efficacy of blood-brain barrier/proteolytic pathways.

The kinetics of amyloid beta turnover within human brain is still poorly understood. We previously found a dramatic decline in the turnover of Aβ peptides in normal aging. It was not known if brain interstitial fluid/cerebrospinal fluid (ISF/CSF) fluid exchange, CSF turnover, blood-brain barrier function or proteolysis were affected by aging or the presence of β amyloid plaques.

SILK studies - capturing the turnover of proteins linked to neurodegenerative diseases.

Alzheimer disease (AD) is one of several neurodegenerative diseases characterized by dysregulation, misfolding and accumulation of specific proteins in the CNS. The stable isotope labelling kinetics (SILK) technique is based on generating amino acids labelled with naturally occurring stable (that is, nonradioactive) isotopes of carbon and/or nitrogen. These labelled amino acids can then be incorporated into proteins, enabling rates of protein production and clearance to be determined in vivo and in vitro without the use of radioactive or chemical labels.

Effect of sleep on overnight cerebrospinal fluid amyloid β kinetics.

Sleep disturbances are associated with future risk of Alzheimer disease. Disrupted sleep increases soluble amyloid β, suggesting a mechanism for sleep disturbances to increase Alzheimer disease risk. We tested this response in humans using indwelling lumbar catheters to serially sample cerebrospinal fluid while participants were sleep-deprived, treated with sodium oxybate, or allowed to sleep normally.

Associations Between β-Amyloid Kinetics and the β-Amyloid Diurnal Pattern in the Central Nervous System.

Importance: Recent studies found that the concentration of amyloid-β (Aβ) fluctuates with the sleep-wake cycle. Although the amplitude of this day/night pattern attenuates with age and amyloid deposition, to our knowledge, the association of Aβ kinetics (ie, production, turnover, and clearance) with this oscillation has not been studied.

Objective: To determine the association between Aβ kinetics, age, amyloid levels, and the Aβ day/night pattern in humans.

A modular, plasmin-sensitive, clickable poly(ethylene glycol)-heparin-laminin microsphere system for establishing growth factor gradients in nerve guidance conduits.

Peripheral nerve regeneration is a complex problem that, despite many advancements and innovations, still has sub-optimal outcomes. Compared to biologically derived acellular nerve grafts and autografts, completely synthetic nerve guidance conduits (NGC), which allow for precise engineering of their properties, are promising but still far from optimal. We have developed an almost entirely synthetic NGC that allows control of soluble growth factor delivery kinetics, cell-initiated degradability and cell attachment.

Age and amyloid effects on human central nervous system amyloid-beta kinetics.

Objective: Age is the single greatest risk factor for Alzheimer's disease (AD), with the incidence doubling every 5 years after age 65. However, our understanding of the mechanistic relationship between increasing age and the risk for AD is currently limited. We therefore sought to determine the relationship between age, amyloidosis, and amyloid-beta (Aβ) kinetics in the central nervous system (CNS) of humans.

Analysis of a compartmental model of amyloid beta production, irreversible loss and exchange in humans.

Amyloid beta (Aβ) peptides, and in particular Aβ42, are found in senile plaques associated with Alzheimer's disease. A compartmental model of Aβ production, exchange and irreversible loss was recently developed to explain the kinetics of isotope-labeling of Aβ peptides collected in cerebrospinal fluid (CSF) following infusion of stable isotope-labeled leucine in humans. The compartmental model allowed calculation of the rates of production, irreversible loss (or turnover) and short-term exchange of Aβ peptides.

Amyloid-β efflux from the central nervous system into the plasma.

Objective: The aim of this study was to measure the flux of amyloid-β (Aβ) across the human cerebral capillary bed to determine whether transport into the blood is a significant mechanism of clearance for Aβ produced in the central nervous system (CNS).

Methods: Time-matched blood samples were simultaneously collected from a cerebral vein (including the sigmoid sinus, inferior petrosal sinus, and the internal jugular vein), femoral vein, and radial artery of patients undergoing inferior petrosal sinus sampling. For each plasma sample, Aβ concentration was assessed by 3 assays, and the venous to arterial Aβ concentration ratios were determined.

Controlled release and gradient formation of human glial-cell derived neurotrophic factor from heparinated poly(ethylene glycol) microsphere-based scaffolds.

Introduction of spatial patterning of proteins, while retaining activity and releasability, is critical for the field of regenerative medicine. Reversible binding to heparin, which many biological molecules exhibit, is one potential pathway to achieve this goal. We have covalently bound heparin to poly(ethylene glycol) (PEG) microspheres to create useful spatial patterns of glial-cell derived human neurotrophic factor (GDNF) in scaffolds to promote peripheral nerve regeneration.

Clickable Poly(ethylene glycol)-Microsphere-Based Cell Scaffolds.

Clickable poly(ethylene glycol) (PEG) derivatives are used with two sequential aqueous two-phase systems to produce microsphere-based scaffolds for cell encapsulation. In the first step, sodium sulfate causes phase separation of the clickable PEG precursors and is followed by rapid geleation to form microspheres in the absence of organic solvent or surfactant. The microspheres are washed and then deswollen in dextran solutions in the presence of cells, producing tightly packed scaffolds that can be easily handled while also maintaining porosity.

Direct reprogramming of mouse fibroblasts to cardiomyocyte-like cells using Yamanaka factors on engineered poly(ethylene glycol) (PEG) hydrogels.

Direct reprogramming strategies enable rapid conversion of somatic cells to cardiomyocytes or cardiomyocyte-like cells without going through the pluripotent state. A recently described protocol couples Yamanaka factor induction with pluripotency inhibition followed by BMP4 treatment to achieve rapid reprogramming of mouse fibroblasts to beating cardiomyocyte-like cells. The original study was performed using Matrigel-coated tissue culture polystyrene (TCPS), a stiff material that also non-specifically adsorbs serum proteins.

Increased in vivo amyloid-β42 production, exchange, and loss in presenilin mutation carriers.

Alzheimer's disease (AD) is hypothesized to be caused by an overproduction or reduced clearance of amyloid-β (Aβ) peptide. Autosomal dominant AD (ADAD) caused by mutations in the presenilin (PSEN) gene have been postulated to result from increased production of Aβ42 compared to Aβ40 in the central nervous system (CNS). This has been demonstrated in rodent models of ADAD but not in human mutation carriers.

Ultralow protein adsorbing coatings from clickable PEG nanogel solutions: benefits of attachment under salt-induced phase separation conditions and comparison with PEG/albumin nanogel coatings.

Clickable nanogel solutions were synthesized by using the copper catalyzed azide/alkyne cycloaddition (CuAAC) to partially polymerize solutions of azide and alkyne functionalized poly(ethylene glycol) (PEG) monomers. Coatings were fabricated using a second click reaction: a UV thiol-yne attachment of the nanogel solutions to mercaptosilanated glass. Because the CuAAC reaction was effectively halted by the addition of a copper-chelator, we were able to prevent bulk gelation and limit the coating thickness to a single monolayer of nanogels in the absence of the solution reaction.

Exposure of the lysine in the gamma chain dodecapeptide of human fibrinogen is not enhanced by adsorption to poly(ethylene terephthalate) as measured by biotinylation and mass spectrometry.

Conformational changes in adsorbed fibrinogen may enhance the exposure of platelet adhesive sites that are inaccessible in solution. To test this hypothesis, mass spectrometric methods were developed to quantify chemical modification of lysine residues following adsorption of fibrinogen to biomaterials. The quantitative method used an internal standard consisting of isotope-labeled fibrinogen secreted by human HepG2 cells in culture.

Long-term culture of HL-1 cardiomyocytes in modular poly(ethylene glycol) microsphere-based scaffolds crosslinked in the phase-separated state.

Poly(ethylene glycol) (PEG) microspheres were assembled around HL-1 cardiomyocytes to produce highly porous modular scaffolds. In this study we took advantage of the immiscibility of PEG and dextran to improve upon our previously described modular scaffold fabrication methods. Phase separating the PEG microspheres in dextran solutions caused them to rapidly deswell and crosslink together, eliminating the need for serum protein-based crosslinking.

Changes of chondrocyte expression profiles in human MSC aggregates in the presence of PEG microspheres and TGF-β3.

Biomaterial microparticles are commonly utilized as growth factor delivery vehicles to induce chondrogenic differentiation of mesenchymal stem/stromal cells (MSCs). To address whether the presence of microparticles could themselves affect differentiation of MSCs, a 3D co-aggregate system was developed containing an equal volume of human primary bone marrow-derived MSCs and non-degradable RGD-conjugated poly(ethylene glycol) microspheres (PEG-μs). Following TGF-β3 induction, differences in cell phenotype, gene expression and protein localization patterns were found when compared to MSC aggregate cultures devoid of PEG-μs.

Modular poly(ethylene glycol) scaffolds provide the ability to decouple the effects of stiffness and protein concentration on PC12 cells.

This research focused on developing a modular poly(ethylene glycol) (PEG) scaffold, assembled from PEG microgels and collagen I, to provide an environment to decouple the chemical and mechanical cues within a three-dimensional scaffold. We first characterized the microgel fabrication process, examining the size, polydispersity, swelling ratio, mesh size and storage modulus of the polymer particles. The resulting microgels had a low polydispersity index, PDI=1.

Bottom-up tissue engineering.

Recapitulating the elegant structures formed during development is an extreme synthetic and biological challenge. Great progress has been made in developing materials to support transplanted cells, yet the complexity of tissues is far beyond that found in even the most advanced scaffolds. Self-assembly is a motif used in development and a route for the production of complex materials.

Poly(ethylene glycol) microparticles produced by precipitation polymerization in aqueous solution.

Methods were developed to perform precipitation photopolymerization of PEG-diacrylate. Previously, comonomers have been added to PEG when precipitation polymerization was desired. In the present method, the LCST of the PEG itself was lowered by the addition of the kosmotropic salt sodium sulfate to an aqueous solution.

Nanogel surface coatings for improved single-molecule imaging substrates.

Surfaces that resist protein adsorption are important for many bioanalytical applications. Bovine serum albumin (BSA) coatings and multi-arm poly(ethylene glycol) (PEG) coatings display low levels of non-specific protein adsorption and have enabled highly quantitative single-molecule (SM) protein studies. Recently, a method was developed for coating a glass with PEG-BSA nanogels, a promising hybrid of these two low-background coatings.

The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres.

A critical element in the formation of scaffolds for tissue engineering is the introduction of concentration gradients of bioactive molecules. We explored the use of poly(ethylene glycol) (PEG) microspheres fabricated via a thermally induced phase separation to facilitate the creation of gradients in scaffolds. PEG microspheres were produced with different densities (buoyancies) and centrifuged to develop microsphere gradients.

Liquid-liquid two-phase systems for the production of porous hydrogels and hydrogel microspheres for biomedical applications: A tutorial review.

Macroporous hydrogels may have direct applications in regenerative medicine as scaffolds to support tissue formation. Hydrogel microspheres may be used as drug-delivery vehicles or as building blocks to assemble modular scaffolds. A variety of techniques exist to produce macroporous hydrogels and hydrogel microspheres.

Prediction of sphingosine 1-phosphate-stimulated endothelial cell migration rates using biochemical measurements.

The ability to predict endothelial cell migration rates may aid in the design of biomaterials that endothelialize following implantation. However, the complexity of the signaling response to migration-promoting stimuli such as sphingosine 1-phosphate (S1P) makes such predictions quite challenging. A number of signaling pathways impact S1P-mediated cell migration, including the Akt and Src pathways, which both affect activation of the small GTPase Rac.