Exploring Mechanistic Toxicity of Mixtures Using PBPK Modeling and Computational Systems Biology.

Mixtures risk assessment needs an efficient integration of in vivo, in vitro, and in silico data with epidemiology and human studies data. This involves several approaches, some in current use and others under development. This work extends the Agency for Toxic Substances and Disease Registry physiologically based pharmacokinetic (PBPK) toolkit, available for risk assessors, to include a mixture PBPK model of benzene, toluene, ethylbenzene, and xylenes. The recoded model was evaluated and applied to exposure scenarios to evaluate the validity of dose additivity for mixtures. In the second part of this work, we studied toluene, ethylbenzene, and xylene (TEX)-gene-disease associations using Comparative Toxicogenomics Database, pathway analysis and published microarray data from human gene expression changes in blood samples after short- and long-term exposures. Collectively, this information was used to establish hypotheses on potential linkages between TEX exposures and human health. The results show that 236 genes expressed were common between the short- and long-term exposures. These genes could be central for the interconnecting biological pathways potentially stimulated by TEX exposure, likely related to respiratory and neuro diseases. Using publicly available data we propose a conceptual framework to study pathway perturbations leading to toxicity of chemical mixtures. This proposed methodology lends mechanistic insights of the toxicity of mixtures and when experimentally validated will allow data gaps filling for mixtures' toxicity assessment. This work proposes an approach using current knowledge, available multiple stream data and applying computational methods to advance mixtures risk assessment.

Distinct ON/OFF fluorescence signals from dual-responsive activatable nanoprobes allows detection of inflammation with improved contrast.

Visualization of biochemical changes associated with disease is of great clinical significance, as it should allow earlier, more accurate diagnosis than structural imaging, facilitating timely clinical intervention. Herein, we report combining stimuli-responsive polymers and near-infrared fluorescent dyes (emission max: 790 nm) to create robust activatable fluorescent nanoprobes capable of simultaneously detecting acidosis and oxidative stress associated with inflammatory microenvironments. The spectrally-resolved mechanism of fluorescence activation allows removal of unwanted background signal (up to 20-fold reduction) and isolation of a pure activated signal, which enables sensitive and unambiguous localization of inflamed areas; target-to-background ratios reach 22 as early as 3 h post-injection. This new detection platform could have significant clinical impact in early detection of pathologies, individual tailoring of drug therapy, and image-guided tumor resection.

High throughput bioassay for beta1-adrenoceptor autoantibody detection.

BACKGROUND: While the involvement of adrenergic beta1-autoantibodies (beta1-AABs) in pathogenesis of cardiomyopathies is well established as are the benefits associated with autoantibody removal by immunoapheresis, the development of drugs neutralizing beta1-AABs in-vivo has been slowed due to a lack of high throughput autoantibody analytics. Highly scalable routine diagnostics involving immobilized binding partners have mostly failed in comparison to the laborious bioassays, which are difficult to scale up, but present the most reliable and sensitive tools for detecting the beta1-autoantibodies. METHODS: A high throughput, image-based assay to measure cardiomyocyte beat rate and contractility was developed and tested for its applicability for detecting adrenergic beta1-autoantibodies. The classical bioassay of spontaneously beating neonatal rat cardiomyocytes was used for comparison. RESULTS: The high throughout assay using human iPSC-derived cardiomyocytes was able to detect beta1-AAB activity of biological sample material. The results from the high throughput assay were very similar to the data obtained from the original bioassay of spontaneously beating neonatal cardiomyocytes, with one exception, where a control antibody targeting the N-terminal end of the human beta1-receptor induced a response when tested with the high throughput imager, while none was observed by the classical bioassay. This discrepancy may be explained by the differences in host species of cardiomyocytes tested by the two methods. CONCLUSION: The high throughput system using iPSC-derived cardiomyocytes for the detection of beta1-AAB provides a realistic option to overcome the sample-size limitations of the bioassay-based diagnostics.

Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction.

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.

Prion transmission prevented by modifying the ß2-α2 loop structure of host PrPC.

Zoonotic prion transmission was reported after the bovine spongiform encephalopathy (BSE) epidemic, when >200 cases of prion disease in humans were diagnosed as variant Creutzfeldt-Jakob disease. Assessing the risk of cross-species prion transmission remains challenging. We and others have studied how specific amino acid residue differences between species impact prion conversion and have found that the ß2-α2 loop region of the mouse prion protein (residues 165-175) markedly influences infection by sheep scrapie, BSE, mouse-adapted scrapie, deer chronic wasting disease, and hamster-adapted scrapie prions. The tyrosine residue at position 169 is strictly conserved among mammals and an aromatic side chain in this position is essential to maintain a 310-helical turn in the ß2-α2 loop. Here we examined the impact of the Y169G substitution together with the previously described S170N, N174T "rigid loop" substitutions on cross-species prion transmission in vivo and in vitro. We found that transgenic mice expressing mouse PrP containing the triple-amino acid substitution completely resisted infection with two strains of mouse prions and with deer chronic wasting disease prions. These studies indicate that Y169 is important for prion formation, and they provide a strong indication that variation of the ß2-α2 loop structure can modulate interspecies prion transmission.

Exploiting oxidative microenvironments in the body as triggers for drug delivery systems.

SIGNIFICANCE: Reactive oxygen species and reactive nitrogen species (ROS/RNS) play an important role in cell signaling pathways. However, the increased production of these species may disrupt cellular homeostasis, giving rise to pathological conditions. Biomaterials that are responsive to ROS/RNS can be strategically used to specifically release therapeutics and diagnostic agents to regions undergoing oxidative stress. RECENT ADVANCES: Many nanocarriers intended to exploit redox micro-environments as triggers for drug release, summarized and compared in this review, have recently been developed. We describe these carriers' chemical structures, strategies for payload protection and oxidation-selective release, and ROS/RNS sensitivity as tested in initial studies. CRITICAL ISSUES: ROS/RNS are unstable, so reliable measures of their concentrations in various conditions are scarce. Combined with the dearth of materials shown to respond to physiologically relevant levels of ROS/RNS, evaluations of their true sensitivity are difficult. FUTURE DIRECTIONS: Oxidation-responsive nanocarriers developed thus far show tremendous potential for applicability in vivo; however, the sensitivity of these chemistries needs to be fine tuned to enable responses to physiological levels of ROS and RNS.

De novo prion aggregates trigger autophagy in skeletal muscle.

In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

Density gradient multilayered polymerization (DGMP): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering.

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer. Current methodologies to achieve this include photopatterning, lithography, sequential functionalization5, freeze drying, microfluidics, or centrifugation, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers. DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials. A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications.

A Single UV or Near IR Triggering Event Leads to Polymer Degradation into Small Molecules.

We report two polymers with UV- and NIR-removable end caps that respond to a single light activated event by complete cleavage of the polymer backbone via a self-immolative mechanism. Two photocleavable protecting groups were used to cap the polymers; o-nitrobenzyl alcohol (ONB) and bromo-coumarin (Bhc). GPC and (1)H NMR confirmed complete degradation of the ONB-containing polymer in response to UV. The polymers were formulated into nanoparticles; fluorescence measurements of encapsulated Nile red confirmed release upon photolysis of the endcaps. Contrary to previous work using a similar backbone structure that degrades upon hydrolysis, here, the disassembly process and burst release of the payload are only activated on demand, illustrating the powerful capacity of light to trigger release from polymeric nanoparticles. Our design allows the signal to be amplified in a domino effect to fully degrade the polymer into small molecules. Thus, polymers and nanoparticles can reach maximal degradation without having to use intense and/or long periods of irradiation.

Biocompatible polymeric nanoparticles degrade and release cargo in response to biologically relevant levels of hydrogen peroxide.

Oxidative stress is caused predominantly by accumulation of hydrogen peroxide and distinguishes inflamed tissue from healthy tissue. Hydrogen peroxide could potentially be useful as a stimulus for targeted drug delivery to diseased tissue. However, current polymeric systems are not sensitive to biologically relevant concentrations of H(2)O(2) (50-100 µM). Here we report a new biocompatible polymeric capsule capable of undergoing backbone degradation and thus release upon exposure to such concentrations of hydrogen peroxide. Two polymeric structures were developed differing with respect to the linkage between the boronic ester group and the polymeric backbone: either direct (1) or via an ether linkage (2). Both polymers are stable in aqueous solution at normal pH, and exposure to peroxide induces the removal of the boronic ester protecting groups at physiological pH and temperature, revealing phenols along the backbone, which undergo quinone methide rearrangement to lead to polymer degradation. Considerably faster backbone degradation was observed for polymer 2 over polymer 1 by NMR and GPC. Nanoparticles were formulated from these novel materials to analyze their oxidation triggered release properties. While nanoparticles formulated from polymer 1 only released 50% of the reporter dye after exposure to 1 mM H(2)O(2) for 26 h, nanoparticles formulated from polymer 2 did so within 10 h and were able to release their cargo selectively in biologically relevant concentrations of H(2)O(2). Nanoparticles formulated from polymer 2 showed a 2-fold enhancement of release upon incubation with activated neutrophils, while controls showed a nonspecific response to ROS producing cells. These polymers represent a novel, biologically relevant, and biocompatible approach to biodegradable H(2)O(2)-triggered release systems that can degrade into small molecules, release their cargo, and should be easily cleared by the body.

Biochemical properties of highly neuroinvasive prion strains.

Infectious prions propagate from peripheral entry sites into the central nervous system (CNS), where they cause progressive neurodegeneration that ultimately leads to death. Yet the pathogenesis of prion disease can vary dramatically depending on the strain, or conformational variant of the aberrantly folded and aggregated protein, PrP(Sc). Although most prion strains invade the CNS, some prion strains cannot gain entry and do not cause clinical signs of disease. The conformational basis for this remarkable variation in the pathogenesis among strains is unclear. Using mouse-adapted prion strains, here we show that highly neuroinvasive prion strains primarily form diffuse aggregates in brain and are noncongophilic, conformationally unstable in denaturing conditions, and lead to rapidly lethal disease. These neuroinvasive strains efficiently generate PrP(Sc) over short incubation periods. In contrast, the weakly neuroinvasive prion strains form large fibrillary plaques and are stable, congophilic, and inefficiently generate PrP(Sc) over long incubation periods. Overall, these results indicate that the most neuroinvasive prion strains are also the least stable, and support the concept that the efficient replication and unstable nature of the most rapidly converting prions may be a feature linked to their efficient spread into the CNS.

Spongiform encephalopathy in transgenic mice expressing a point mutation in the ß2-α2 loop of the prion protein.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrP(C), into a ß-sheet-rich, aggregated isoform, PrP(Sc). We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the ß2-α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered ß2-α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrP(D167S) developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the ß2-α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.

Prion strain interactions are highly selective.

Various misfolded and aggregated neuronal proteins commonly coexist in neurodegenerative disease, but whether the proteins coaggregate and alter the disease pathogenesis is unclear. Here, we used mixtures of distinct prion strains, which are believed to differ in conformation, to test the hypothesis that two different aggregates interact and change the disease in vivo. We tracked two prion strains in mice histopathologically and biochemically, as well as by spectral analysis of plaque-bound PTAA (polythiophene acetic acid), a conformation-sensitive fluorescent amyloid ligand. We found that prion strains interacted in a highly selective and strain-specific manner, with (1) no interaction, (2) hybrid plaque formation, or (3) blockage of one strain by a second (interference). The hybrid plaques were maintained on additional passage in vivo and each strain seemed to maintain its original conformational properties, suggesting that one strain served only as a scaffold for aggregation of the second strain. These findings not only further our understanding of prion strain interactions but also directly demonstrate interactions that may occur in other protein aggregate mixtures.