Ataxin-3 Regulates Aggresome Formation of Copper-Zinc Superoxide Dismutase (SOD1) by Editing K63-linked Polyubiquitin Chains

Polyubiquitination of misfolded proteins, especially K63-linked polyubiquitination, is thought to be associated with the formation of inclusion bodies. However, it is not well explored whether appropriate editing of the different types of ubiquitin linkages by deubiquitinating enzymes (DUBs) affects the dynamics of inclusion bodies. In this study, we report that a specific DUB, ataxin-3, is required for the efficient recruitment of the neurodegenerative disease-associated protein copper-zinc superoxide dismutase (SOD1) to aggresomes. The overexpression of ataxin-3 promotes mutant SOD1 aggresome formation by trimming K63-linked polyubiquitin chains. Moreover, knockdown of ataxin-3 decreases mutant SOD1 aggresome formation and increases cell death induced by mutant SOD1. Thus, our data suggest that the sequestration of misfolded SOD1 into aggresomes, which is driven by ataxin-3, plays an important role in attenuating protein misfolding-induced cell toxicity.     

Observer-based tracking control of abnormal oscillations in demyelination symptom

Fast axonal conduction of action potentials in mammals relies on myelin insulation. Demyelination can cause slowed, blocked, desynchronized, or paradoxically excessive spiking that underlies the symptoms observed in demyelination diseases. Feedback control via functional electrical stimulation (FES) seems to be a promising treatment modality in such diseases. However, there are challenges to implementing such method for neurons: high nonlinearity, biological tissue constrains and unobservable ion channel states. To address this problem, we propose an estimating and tracking control strategy for systems based on Kalman filter, in order to enhance the action potential propagation reliability of demyelinated neuron via FES. Unscented Kalman filter (UKF) is employed to estimate the unobservable states and parameters in the demyelination neuron model from membrane potential dynamics. Our method could promote the design of new closed-loop electrical stimulation systems for patients suffering from different nerve system dysfunctions.     

Outcomes in Cochrane Systematic Reviews Addressing Four Common Eye Conditions: An Evaluation of Completeness and Comparability

Introduction

Choice of outcomes is critical for clinical trialists and systematic reviewers. It is currently unclear how systematic reviewers choose and pre-specify outcomes for systematic reviews. Our objective was to assess the completeness of pre-specification and comparability of outcomes in all Cochrane reviews addressing four common eye conditions.

Methods

We examined protocols for all Cochrane reviews as of June 2013 that addressed glaucoma, cataract, age-related macular degeneration (AMD), and diabetic retinopathy (DR). We assessed completeness and comparability for each outcome that was named in ≥25% of protocols on those topics. We defined a completely-specified outcome as including information about five elements: domain, specific measurement, specific metric, method of aggregation, and time-points. For each domain, we assessed comparability in how individual elements were specified across protocols.

Results

We identified 57 protocols addressing glaucoma (22), cataract (16), AMD (15), and DR (4). We assessed completeness and comparability for five outcome domains: quality-of-life, visual acuity, intraocular pressure, disease progression, and contrast sensitivity. Overall, these five outcome domains appeared 145 times (instances). Only 15/145 instances (10.3%) were completely specified (all five elements) (median = three elements per outcome). Primary outcomes were more completely specified than non-primary (median = four versus two elements). Quality-of-life was least completely specified (median = one element). Due to largely incomplete outcome pre-specification, conclusive assessment of comparability in outcome usage across the various protocols per condition was not possible.

Discussion

Outcome pre-specification was largely incomplete; we encourage systematic reviewers to consider all five elements. This will indicate the importance of complete specification to clinical trialists, on whose work systematic reviewers depend, and will indirectly encourage comparable outcome choice to reviewers undertaking related research questions. Complete pre-specification could improve efficiency and reduce bias in data abstraction and analysis during a systematic review. Ultimately, more completely specified and comparable outcomes could make systematic reviews more useful to decision-makers.     

Association Mapping and Haplotype Analysis of a 3.1-Mb Genomic Region Involved in Fusarium Head Blight Resistance on Wheat Chromosome 3BS

A previous study provided an in-depth understanding of molecular population genetics of European and Asian wheat gene pools using a sequenced 3.1-Mb contig (ctg954) on chromosome 3BS. This region is believed to carry the Fhb1 gene for response to Fusarium head blight. In this study, 266 wheat accessions were evaluated in three environments for Type II FHB response based on the single floret inoculation method. Hierarchical clustering (UPGMA) based on a Manhattan dissimilarity matrix divided the accessions into eight groups according to five FHB-related traits which have a high correlation between them; Group VIII comprised six accessions with FHB response levels similar to variety Sumai 3. Based on the compressed mixed linear model (MLM), association analysis between five FHB-related traits and 42 molecular markers along the 3.1-Mb region revealed 12 significant association signals at a threshold of P<0.05. The highest proportion of phenotypic variation (6.2%) in number of diseased spikelets (NDS) occurred at locus cfb6059, and the physical distance was about 2.9 Kb between umn10 and this marker. Haplotype block (HapB) analysis using a sliding window LD of 5 markers, detected six HapBs in the 3.1-Mb region at r²>0.1 and P<0.001 between random closely linked markers. F-tests among Haps with frequencies >0.05 within each HapB at r²>0.1 and P<0.001 showed significant differences between the Hap carried by FHB resistant resources, such as Sumai 3 and Wangshuibai, and susceptible genotypes in HapB3 and HapB6. These results suggest that Fhb1 is located within HapB6, with the possibility that another gene is located at or near HapB3. SSR markers and Haps detected in this study will be helpful in further understanding the genetic basis of FHB resistance, and provide useful information for marker-assisted selection of Fhb1 in wheat breeding.     

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N^ω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.     

Suppression of FUT1/FUT4 expression by siRNA inhibits tumor growth

Lewis Y (LeY) antigen is highly expressed in a variety of human carcinomas of epithelial cell origin. Recent studies suggest functional blockade of LeY may provide a novel therapeutic approach for the treatment of cancers. However, suppressing LeY expression by genetic manipulation and its impact on neoplastic cell proliferation has not been investigated. We report here that different fucosyltransferases (FUTs) were expressed with the greatest expression of fucosyltransferase I or IV (FUT1/4), the two key enzymes for the synthesis of LeY in human epidermoid carcinoma A431 cells. Knocking down FUT1/4 expression by short interfering RNA technique dramatically reduced the expression of FUT1/4 and LeY and inhibited cell proliferation through decreasing epidermal growth factor receptor (EGFR) signaling pathway. Treatment of A431 cells that were inoculated into the nude mice with FUT1 siRNA or FUT4 siRNA greatly impeded tumor growth. Suppressing FUT1/4 expression also blocked EGF-induced tyrosine phosphorylation of EGFR and mitogen-activated protein kinases. In conclusion, suppressing the expression of FUT1/4 by RNAi technology reduces the synthesis of LeY and inhibits cancer growth. It may serve as a potential methodology for the treatment of cancers that express LeY glycoconjugates.     

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology^1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry⁵ (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells^7-10.Download video file.^{(41M, mov)}