Experimental and Computational Analysis of Polyglutamine-Mediated Cytotoxicity

Expanded polyglutamine (polyQ) proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. PolyQ tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyQ into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of polyQ, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyQ protein inclusion, body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death

Optical Fibre Based Real-Time Measurements During an LDR Prostate Brachytherapy Implant Simulation: Using a 3D printed anthropomorphic phantom

An optical fbre sensor based on radioluminescence, using the scintillation material terbium doped gadolinium oxysulphide (Gd2O2S:Tb) is evaluated, using a 3D printed anthropomorphic phantom for applications in low dose-rate (LDR) prostate brachytherapy. The scintillation material is embedded in a 700 µm diameter cavity within a 1 mm plastic optical fbre that is fxed within a brachytherapy needle. The high spatial resolution dosimeter is used to measure the dose contribution from Iodine-125 (I-125) seeds. Initially, the efects of sterilisation on the sensors (1) repeatability, (2) response as a function of angle, and (3) response as a function of distance, are evaluated in a custom polymethyl methacrylate phantom. Results obtained in this study demonstrate that the output response of the sensor, pre- and post-sterilisation are within the acceptable measurement uncertainty ranging from a maximum standard deviation of 4.7% pre and 5.5% post respectively, indicating that the low temperature sterilisation process does not damage the sensor or reduce performance. Subsequently, an LDR brachytherapy plan reconstructed using the VariSeed treatment planning system, in an anthropomorphic 3D printed training phantom, was used to assess the suitability of the sensor for applications in LDR brachytherapy. This phantom was printed based on patient anatomy, with the volume and dimensions of the prostate designed to represent that of the patient. I-125 brachytherapy seeds, with an average activity of 0.410 mCi, were implanted into the prostate phantom under transrectal ultrasound guidance; following the same techniques as employed in clinical practice by an experienced radiation oncologist. This work has demonstrated that this sensor is capable of accurately identifying when radioactive I-125 sources are introduced into the prostate via a brachytherapy needle

Loss of UCHL1 promotes age-related degenerative changes in the enteric nervous system.

UCHL1 (ubiquitin carboxyterminal hydrolase 1) is a deubiquitinating enzyme that is particularly abundant in neurons. From studies of a spontaneous mutation arising in a mouse line it is clear that loss of function of UCHL1 generates profound degenerative changes in the central nervous system, and it is likely that a proteolytic deficit contributes to the pathology. Here these effects were found to be recapitulated in mice in which the Uchl1 gene had been inactivated by homologous recombination. In addition to the previously documented neuropathology associated with loss of UCHL1 function, axonal swellings were detected in the striatum. In agreement with previously reported findings the loss of UCHL1 function was accompanied by perturbations in ubiquitin pools, but glutathione levels were also significantly depleted in the brains of the knockout mice, suggesting that oxidative defense mechanisms may be doubly compromised. To determine if, in addition to its role in the central nervous system, UCHL1 function is also required for homeostasis of the enteric nervous system the gastrointestinal tract was analyzed in UCHL1 knockout mice. The mice displayed functional changes and morphological changes in gut neurons that preceded degenerative changes in the brain. The changes were qualitatively and quantitatively similar to those observed in wild type mice of much greater age, and strongly resemble changes reported for elderly humans. UCHL1 knockout mice should therefore serve as a useful model of gut aging

Prion protein amyloidosis with divergent phenotype associated with two novel nonsense mutations in PRNP

Stop codon mutations in the gene encoding the prion protein (PRNP) are very rare and have thus far only been described in two patients with prion protein cerebral amyloid angiopathy (PrP-CAA). In this report, we describe the clinical, histopathological and pathological prion protein (PrPSc) characteristics of two Dutch patients carrying novel adjacent stop codon mutations in the C-terminal part of PRNP, resulting in either case in hereditary prion protein amyloidoses, but with strikingly different clinicopathological phenotypes. The patient with the shortest disease duration (27 months) carried a Y226X mutation and showed PrP-CAA without any neurofibrillary lesions, whereas the patient with the longest disease duration (72 months) had a Q227X mutation and showed an unusual Gerstmann-Sträussler-Scheinker disease phenotype with numerous cerebral multicentric amyloid plaques and severe neurofibrillary lesions without PrP-CAA. Western blot analysis in the patient with the Q227X mutation demonstrated the presence of a 7 kDa unglycosylated PrPSc fragment truncated at both the N- and C-terminal ends. Our observations expand the spectrum of clinicopathological phenotypes associated with PRNP mutations and show that a single tyrosine residue difference in the PrP C-terminus may significantly affect the site of amyloid deposition and the overall phenotypic expression of the prion disease. Furthermore, it confirms that the absence of the glycosylphosphatidylinositol anchor in PrP predisposes to amyloid plaque formation

Contribution of Chondroitin Sulfate A to the Binding of Complement Proteins to Activated Platelets

Exposure of chondroitin sulfate A (CS-A) on the surface of activated platelets is well established. The aim of the present study was to investigate to what extent CS-A contributes to the binding of the complement recognition molecule C1q and the complement regulators C1 inhibitor (C1INH), C4b-binding protein (C4BP), and factor H to platelets.Human blood serum was passed over Sepharose conjugated with CS-A, and CS-A-specific binding proteins were identified by Western blotting and mass spectrometric analysis. C1q was shown to be the main protein that specifically bound to CS-A, but C4BP and factor H were also shown to interact. Binding of C1INH was dependent of the presence of C1q and then not bound to CS-A from C1q-depleted serum. The specific interactions observed of these proteins with CS-A were subsequently confirmed by surface plasmon resonance analysis using purified proteins. Importantly, C1q, C4BP, and factor H were also shown to bind to activated platelets and this interaction was inhibited by a CS-A-specific monoclonal antibody, thereby linking the binding of C1q, C4BP, and factor H to exposure of CS-A on activated platelets. CS-A-bound C1q was also shown to amplify the binding of model immune complexes to both microtiter plate-bound CS-A and to activated platelets.This study supports the concept that CS-A contributes to the binding of C1q, C4BP, and factor H to platelets, thereby adding CS-A to the previously reported binding sites for these proteins on the platelet surface. CS-A-bound C1q also seems to amplify the binding of immune complexes to activated platelets, suggesting a role for this molecule in immune complex diseases

Extent and patterns of community collaboration in local health departments: An exploratory survey

<p>Abstract</p> <p>Background</p> <p>Local public health departments (LHDs) in the United States have been encouraged to collaborate with various other community organizations and individuals. Current research suggests that many forms of active partnering are ongoing, and there are numerous examples of LHD collaboration with a specific organization for a specific purpose or program. However, no existing research has attempted to characterize collaboration, for the defined purpose of setting community health status priorities, between a defined population of local officials and a defined group of alternative partnering organizations. The specific aims of this study were to 1) determine the range of collaborative involvement exhibited by a study population of local public health officials, and, 2) characterize the patterns of the selection of organizations/individuals involved with LHDs in the process of setting community health status priorities.</p> <p>Methods</p> <p>Local health department officials in North Carolina (n = 53) responded to an exploratory survey about their levels of involvement with eight types of possible collaborator organizations and individuals. Descriptive statistics and the stochastic clustering technique of Self-Organizing Maps (SOM) were used to characterize their collaboration.</p> <p>Results</p> <p>Local health officials vary extensively in their level of collaboration with external collaborators. While the range of total involvement varies, the patterns of involvement for this specific function are relatively uniform. That is, regardless of the total level of involvement (low, medium or high), officials maintain similar hierarchical preference rankings with Community Advisory Boards and Local Boards of Health most involved and Experts and Elected Officials least involved.</p> <p>Conclusion</p> <p>The extent and patterns of collaboration among LHDs with other community stakeholders for a specific function can be described and ultimately related to outcome measures of LHD performance.</p

Entrapment of Viral Capsids in Nuclear PML Cages Is an Intrinsic Antiviral Host Defense against Varicella-Zoster Virus

The herpesviruses, like most other DNA viruses, replicate in the host cell nucleus. Subnuclear domains known as promyelocytic leukemia protein nuclear bodies (PML-NBs), or ND10 bodies, have been implicated in restricting early herpesviral gene expression. These viruses have evolved countermeasures to disperse PML-NBs, as shown in cells infected in vitro, but information about the fate of PML-NBs and their functions in herpesvirus infected cells in vivo is limited. Varicella-zoster virus (VZV) is an alphaherpesvirus with tropism for skin, lymphocytes and sensory ganglia, where it establishes latency. Here, we identify large PML-NBs that sequester newly assembled nucleocapsids (NC) in neurons and satellite cells of human dorsal root ganglia (DRG) and skin cells infected with VZV in vivo. Quantitative immuno-electron microscopy revealed that these distinctive nuclear bodies consisted of PML fibers forming spherical cages that enclosed mature and immature VZV NCs. Of six PML isoforms, only PML IV promoted the sequestration of NCs. PML IV significantly inhibited viral infection and interacted with the ORF23 capsid surface protein, which was identified as a target for PML-mediated NC sequestration. The unique PML IV C-terminal domain was required for both capsid entrapment and antiviral activity. Similar large PML-NBs, termed clastosomes, sequester aberrant polyglutamine (polyQ) proteins, such as Huntingtin (Htt), in several neurodegenerative disorders. We found that PML IV cages co-sequester HttQ72 and ORF23 protein in VZV infected cells. Our data show that PML cages contribute to the intrinsic antiviral defense by sensing and entrapping VZV nucleocapsids, thereby preventing their nuclear egress and inhibiting formation of infectious virus particles. The efficient sequestration of virion capsids in PML cages appears to be the outcome of a basic cytoprotective function of this distinctive category of PML-NBs in sensing and safely containing nuclear aggregates of aberrant proteins

Aging-related tau astrogliopathy (ARTAG):harmonized evaluation strategy

Pathological accumulation of abnormally phosphorylated tau protein in astrocytes is a frequent, but poorly characterized feature of the aging brain. Its etiology is uncertain, but its presence is sufficiently ubiquitous to merit further characterization and classification, which may stimulate clinicopathological studies and research into its pathobiology. This paper aims to harmonize evaluation and nomenclature of aging-related tau astrogliopathy (ARTAG), a term that refers to a morphological spectrum of astroglial pathology detected by tau immunohistochemistry, especially with phosphorylation-dependent and 4R isoform-specific antibodies. ARTAG occurs mainly, but not exclusively, in individuals over 60 years of age. Tau-immunoreactive astrocytes in ARTAG include thorn-shaped astrocytes at the glia limitans and in white matter, as well as solitary or clustered astrocytes with perinuclear cytoplasmic tau immunoreactivity that extends into the astroglial processes as fine fibrillar or granular immunopositivity, typically in gray matter. Various forms of ARTAG may coexist in the same brain and might reflect different pathogenic processes. Based on morphology and anatomical distribution, ARTAG can be distinguished from primary tauopathies, but may be concurrent with primary tauopathies or other disorders. We recommend four steps for evaluation of ARTAG: (1) identification of five types based on the location of either morphologies of tau astrogliopathy: subpial, subependymal, perivascular, white matter, gray matter; (2) documentation of the regional involvement: medial temporal lobe, lobar (frontal, parietal, occipital, lateral temporal), subcortical, brainstem; (3) documentation of the severity of tau astrogliopathy; and (4) description of subregional involvement. Some types of ARTAG may underlie neurological symptoms; however, the clinical significance of ARTAG is currently uncertain and awaits further studies. The goal of this proposal is to raise awareness of astroglial tau pathology in the aged brain, facilitating communication among neuropathologists and researchers, and informing interpretation of clinical biomarkers and imaging studies that focus on tau-related indicators