Publications by authors named "Justin Savage"

17 Publications

  • Page 1 of 1

Emerging Role of PD-1 in the Central Nervous System and Brain Diseases.

Neurosci Bull 2021 Apr 20. Epub 2021 Apr 20.

Department of Anesthesiology, Duke University Medical Center, Durham, 27710, USA.

Programmed cell death protein 1 (PD-1) is an immune checkpoint modulator and a major target of immunotherapy as anti-PD-1 monoclonal antibodies have demonstrated remarkable efficacy in cancer treatment. Accumulating evidence suggests an important role of PD-1 in the central nervous system (CNS). PD-1 has been implicated in CNS disorders such as brain tumors, Alzheimer's disease, ischemic stroke, spinal cord injury, multiple sclerosis, cognitive function, and pain. PD-1 signaling suppresses the CNS immune response via resident microglia and infiltrating peripheral immune cells. Notably, PD-1 is also widely expressed in neurons and suppresses neuronal activity via downstream Src homology 2 domain-containing protein tyrosine phosphatase 1 and modulation of ion channel function. An improved understanding of PD-1 signaling in the cross-talk between glial cells, neurons, and peripheral immune cells in the CNS will shed light on immunomodulation, neuromodulation, and novel strategies for treating brain diseases.
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http://dx.doi.org/10.1007/s12264-021-00683-yDOI Listing
April 2021

GM-1111 reduces radiation-induced oral mucositis in mice by targeting pattern recognition receptor-mediated inflammatory signaling.

PLoS One 2021 26;16(3):e0249343. Epub 2021 Mar 26.

GlycoMira Therapeutics, Salt Lake City, Utah, United States of America.

Purpose: Oral mucositis (OM) is a common, painful side effect of radiation therapy used for the treatment of head and neck cancer (HNC). Activation of the innate immune system upon irradiation has been identified as a key precipitating event of OM. To better understand OM's pathogenesis, we studied pattern recognition receptors (PRRs) and their downstream pro-inflammatory cytokines in a mouse model of radiation-induced OM. We also tested therapeutic efficacy of GM-1111 that targets innate immune system to reduce radiation-induced OM.

Methods And Materials: The pathogenesis of OM was studied in a single X-ray induced mouse model. The severity of OM was measured by visual and microscopical examinations. The irradiation-induced changes of PRRs and their downstream effector cytokine gene expression levels were determined. The efficacy of GM-1111 to reduce OM was tested in single and fractionated irradiation mouse models. The impact of the drug on tumor response to radiation therapy was also tested in a mouse model of human HNC.

Results: Radiation-induced tissue ulcerations were radiation-dosage and -time dependent. The lesions showed selective increases in PRR and pro-inflammatory cytokine gene expression levels. Once daily administration of GM-1111 (≥30 mg/kg, s.c.) significantly reduced the severity and the incidence of OM. The drug had little effect on PRRs but significantly inhibited downstream pro-inflammatory cytokine genes. GM-1111 did not interfere radiation therapy to induce HNC SCC-25 tumor regression. Instead, we observed significant drug-induced tumor regression.

Conclusions: Radiation induces tissue damages. The increased expression levels of PRRs and their downstream pro-inflammatory cytokine genes in the damaged tissues suggest their important contribution to the pathogenesis of OM. Drug GM-1111 that targets these innate immune molecules may be a potential drug candidate as an intervention for OM.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0249343PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997003PMC
March 2021

Polysulfated Hyaluronan GlycoMira-1111 Inhibits Elastase and Improves Rheology in Cystic Fibrosis Sputum.

Am J Respir Cell Mol Biol 2021 02;64(2):260-267

Division of Pediatric Pulmonology, Children's Hospital of Richmond at Virginia Commonwealth University, Richmond, Virginia.

Cystic fibrosis (CF) lung disease is marked by high concentrations of neutrophil elastase (NE) and DNA polymers; both factors contribute to airway disease. Although inhaled recombinant human dornase alfa reduces the frequency of CF pulmonary exacerbations, it also increases free NE activity in the sputum. There are no approved anti-NE therapies for patients with CF. We investigated whether synthetic, low-molecular weight polysulfated hyaluronan GlycoMira-1111 (GM-1111) would be effective as an anti-NE drug using CF sputum. Anti-NE activity of GM-1111 was tested in CF sputum in the presence or absence of dornase alfa and/or hypertonic saline using a spectrophotometric assay specific for human NE and was compared with unfractionated heparin. We tested whether GM-1111 disaggregated DNA from CF sputum (using gel electrophoresis analysis) or modified CF sputum viscoelastic properties (using a dynamic rheometer). GM-1111 and unfractionated heparin had near equivalent anti-NE activity in CF sputum in the presence of dornase alfa. Both GM-1111 and unfractionated heparin retained anti-NE activity in hypertonic saline but with decreased activity. GM-1111 increased the release of soluble DNA in CF sputum, resulting in improved depolymerization efficacy of dornase alfa. GM-1111 decreased CF sputum elasticity. GM-1111 inhibited NE activity, enhanced DNA depolymerization by deoxyribonuclease, and decreased viscoelastic properties of CF sputum, similar to effects reported previously for unfractionated heparin. Unlike heparins, GM-1111 is synthetic, with minimal anticoagulant activity, and is not derived from animal products. These key attributes provide advantages over unfractionated heparin as a potential therapeutic for CF.
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http://dx.doi.org/10.1165/rcmb.2020-0157OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874391PMC
February 2021

A synthetic glycosaminoglycan reduces sinonasal inflammation in a murine model of chronic rhinosinusitis.

PLoS One 2018 25;13(9):e0204709. Epub 2018 Sep 25.

Division of Head and Neck Surgery, Rhinology-Sinus and Skull Base Surgery Program, Department of Surgery, University of Utah School of Medicine, Salt Lake City, Utah, United States of America.

Chronic rhinosinusitis (CRS) is characterized by sustained mucosal inflammation, impaired mucociliary clearance, loss of cilia and epithelial barrier breakdown, and tissue remodeling. Certain glycosaminoglycans inhibit various inflammatory mediators, suppress bacterial growth, and provide important functions in mucosal tissue repair and mucociliary clearance. Herein, we evaluated the effects of a synthetic glycosaminoglycan, GM-1111, on the clinical signs and inflammatory tissue changes associated with CRS in mice. CRS was generated by repeated intranasal applications of Aspergillus fumigatus (A. fumigatus) extracts over 4 weeks. Mice were then intranasally administered GM-1111 (600 μg per dose, 5 times a week) or vehicle (phosphate buffered saline, PBS) for an additional 4 weeks while still being given A. fumigatus extracts to maintain a chronic inflammatory environment with acute exacerbations. Clinical signs indicative of sinonasal inflammation were recorded throughout the study. After 9 weeks, whole blood and sinonasal tissues were harvested for hematological, histological, and biochemical examination. The clinical signs, white blood cell counts, tissue markers of sinonasal inflammation, and histological changes caused by A. fumigatus extract administration were compared to the healthy (PBS vehicle) and GM-1111-treated groups (n = 12 per treatment group). Compared to vehicle-treated animals, animals treated with GM-1111 demonstrated significant reductions in clinical signs (p<0.05), degenerative tissue changes, goblet cell hyperplasia, inflammatory cell infiltration (p<0.01), innate immunity- (tlr2, tlr4, myd88, il1b, tnfa, il6, and il12) and adaptive immunity-associated (ccl11, ccl24, ccl5, il4, il5, and il13) cytokine gene expression (p<0.05 to p<0.0001) in sinonasal tissues, and serum IgE levels (p<0.01). Our data suggest that GM-1111 significantly reduces local and systemic effects of CRS-associated sinonasal inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204709PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6155557PMC
March 2019

A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis.

PLoS One 2016 16;11(6):e0157310. Epub 2016 Jun 16.

GlycoMira Therapeutics, Inc. Salt Lake City, UT, 84108, United States of America.

Background: Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis.

Methods: We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts.

Results: (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1-10 ng/mL vs. > 100 μg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism.

Conclusions: We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157310PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911086PMC
July 2017

Investigating the Familial Basis of Heightened Risk-Taking in Adolescents With Conduct Disorder and Their Unaffected Relatives.

Dev Neuropsychol 2016 Jan-Mar;41(1-2):93-106. Epub 2016 Mar 31.

a Academic Unit of Psychology , University of Southampton , Southampton , United Kingdom.

Previous studies have demonstrated increased risk-taking in adolescents with Conduct Disorder (CD) compared with typically developing controls. Increased risk-taking may partly mediate the pathway from genetic or environmental risk to CD. We investigated the familial basis of risk-taking by examining whether the unaffected relatives of CD probands (n = 22) showed heightened risk-taking in a gambling task, in common with affected probands (n = 44). Adolescents with CD were more likely to select risky options than the typically developing controls (n = 37) and unaffected relatives. Our findings confirm the association between CD and increased risk-taking, but suggest that this decision-making style may not have a familial basis.
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http://dx.doi.org/10.1080/87565641.2016.1145223DOI Listing
August 2017

Isolevuglandin-type lipid aldehydes induce the inflammatory response of macrophages by modifying phosphatidylethanolamines and activating the receptor for advanced glycation endproducts.

Antioxid Redox Signal 2015 Jun 18;22(18):1633-45. Epub 2015 Mar 18.

1Division of Clinical Pharmacology, Vanderbilt University at Nashville, Nashville, Tennessee.

Aims: Increased lipid peroxidation occurs in many conditions associated with inflammation. Because lipid peroxidation produces lipid aldehydes that can induce inflammatory responses through unknown mechanisms, elucidating these mechanisms may lead to development of better treatments for inflammatory diseases. We recently demonstrated that exposure of cultured cells to lipid aldehydes such as isolevuglandins (IsoLG) results in the modification of phosphatidylethanolamine (PE). We therefore sought to determine (i) whether PE modification by isolevuglandins (IsoLG-PE) occurred in vivo, (ii) whether IsoLG-PE stimulated the inflammatory responses of macrophages, and (iii) the identity of receptors mediating the inflammatory effects of IsoLG-PE.

Results: IsoLG-PE levels were elevated in plasma of patients with familial hypercholesterolemia and in the livers of mice fed a high-fat diet to induce obesity and hepatosteatosis. IsoLG-PE potently stimulated nuclear factor kappa B (NFκB) activation and expression of inflammatory cytokines in macrophages. The effects of IsoLG-PE were blocked by the soluble form of the receptor for advanced glycation endproducts (sRAGE) and by RAGE antagonists. Furthermore, macrophages derived from the bone marrow of Ager null mice failed to express inflammatory cytokines in response to IsoLG-PE to the same extent as macrophages from wild-type mice.

Innovation: These studies are the first to identify IsoLG-PE as a mediator of macrophage activation and a specific receptor, RAGE, which mediates its biological effects.

Conclusion: PE modification by IsoLG forms RAGE ligands that activate macrophages, so that the increased IsoLG-PE generated by high circulating cholesterol levels or high-fat diet may play a role in the inflammation associated with these conditions.
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http://dx.doi.org/10.1089/ars.2014.6078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485367PMC
June 2015

Salience Not Status: How Category Labels Influence Feature Inference.

Cogn Sci 2015 Sep 28;39(7):1594-621. Epub 2014 Nov 28.

Psychology and Language Sciences, University College London.

Two main uses of categories are classification and feature inference, and category labels have been widely shown to play a dominant role in feature inference. However, the nature of this influence remains unclear, and we evaluate two contrasting hypotheses formalized as mathematical models: the label special-mechanism hypothesis and the label super-salience hypothesis. The special-mechanism hypothesis is that category labels, unlike other features, trigger inference decision making in reference to the category prototypes. This results in a tendency for prototype-compatible inferences because the labels trigger a special mechanism rather than because of any influences they have on similarity evaluation. The super-salience hypothesis assumes that the large label influence is due to their high salience and corresponding impact on similarity without any need for a special mechanism. Application of the two models to a feature inference task based on a family resemblance category structure yields strong support for the label super-salience hypothesis and in particular does not support the need for a special mechanism based on prototypes.
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http://dx.doi.org/10.1111/cogs.12206DOI Listing
September 2015

Prevention of anti-microbial peptide LL-37-induced apoptosis and ATP release in the urinary bladder by a modified glycosaminoglycan.

PLoS One 2013 30;8(10):e77854. Epub 2013 Oct 30.

GlycoMira Therapeutics, Inc. Salt Lake City, Utah, United States of America.

Interstitial cystitis (IC), often referred to in combination with painful bladder syndrome, is a chronic inflammatory disease of the bladder. Current therapies primarily focus on replenishing urothelial glycosaminoglycan (GAG) layer using GAG analogs and managing pain with supportive therapies. However, the elusive etiology of IC and the lack of animal models to study the disease have been major hurdles developing more effective therapeutics. Previously, we showed an increased urinary concentration of antimicrobial peptide LL-37 in spina bifida patients and used LL-37 to develop a mouse model of cystitis that mimics important clinical findings of IC. Here we investigate (1) the molecular mechanism of LL-37 induced cystitis in cultured human urothelial cells and in mice, (2) the protective effects of GM-0111, a modified GAG, within the context of this mechanism, (3) the physiological and molecular markers that correlate with the severity of the inflammation, and (4) the protective effects of several GAGs using these biomarkers in our LL-37 induced cystitis model. We find that LL-37 quickly induces release of ATP and apoptosis in the urothelium. These changes can be inhibited by a chemically-modified GAG, GM-0111. Furthermore, we also find that GAG analogs provide varying degrees of protection against LL-37 challenge in mice. These findings suggest that GM-0111 and possibly GAG molecules prevent the development of cystitis by blocking the apoptosis and the concurrent release of ATP from the urothelium.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0077854PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813730PMC
September 2014

Physiological relevance of LL-37 induced bladder inflammation and mast cells.

J Urol 2013 Oct 9;190(4 Suppl):1596-1602. Epub 2013 Jan 9.

Division of Pediatric Urology (SO, WJ, LMR, LW, ACH, AMH, SM) and Departments of Surgery (SO, WJ, LMR, LW, ACH, AMH, SM), Medicinal Chemistry (JZ, GDP) and Pharmacotherapy (XY) and Center for Therapeutic Biomaterials (JZ, GDP), University of Utah/Primary Children's Medical Center and GlycoMira Therapeutics, L.L.C. (JS, WYL), Salt Lake City, Utah.

Purpose: We established the physiological relevance of LL-37 induced bladder inflammation. We hypothesized that 1) human urinary LL-37 is increased in pediatric patients with spina bifida, 2) LL-37 induced inflammation occurs in our mouse model via urothelial binding and is dose dependent and 3) LL-37 induced inflammation involves mast cells.

Materials And Methods: To test our first hypothesis, we obtained urine samples from 56 pediatric patients with spina bifida and 22 normal patients. LL-37 was measured by enzyme-linked immunosorbent assay. Our second hypothesis was tested in C57Bl/6 mice challenged with 7 LL-37 concentrations intravesically for 1 hour. At 24 hours tissues were examined histologically and myeloperoxidase assay was done to quantitate inflammation. In separate experiments fluorescent LL-37 was instilled and tissues were obtained immediately (time = 0) and at 24 hours (time = 24). To test our final hypothesis, we performed immunohistochemistry for mast cell tryptase and evaluated 5 high power fields per bladder to determine the mean number of mast cells per mm(2).

Results: Urinary LL-37 was 89-fold higher in patients with spina bifida. Mouse LL-37 dose escalation experiments revealed increased inflammation at higher LL-37 concentrations. Fluorescent LL-37 demonstrated global urothelial binding at time = 0 but was not visible at time = 24. Immunohistochemistry for tryptase revealed mast cell infiltration in all tissue layers. At higher concentrations the LL-37 challenge led to significantly greater mast cell infiltration.

Conclusions: Urinary LL-37 was significantly increased in pediatric patients with spina bifida. To our knowledge we report for the first time that LL-37 can elicit profound, dose dependent bladder inflammation involving the urothelium. Finally, inflammation propagation involves mast cells.
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http://dx.doi.org/10.1016/j.juro.2013.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947931PMC
October 2013

Fearlessness in juvenile offenders is associated with offending rate.

Dev Sci 2013 Jan 10;16(1):84-90. Epub 2012 Oct 10.

School of Psychology, Cardiff University, UK.

Poor fear conditioning is a correlate of violent offending in adults, but there is no evidence concerning juvenile offenders. Our aim was to compare emotional learning in juvenile offenders and controls and establish whether crime rate is related to seriousness of emotional learning problems. To this end, emotional learning was assessed in 42 juvenile offenders by measuring skin conductance responding (SCR) during fear conditioning. Compared to controls, juvenile offenders showed lower conditioned SCRs to visual stimuli associated with a subsequent aversive stimulus and the magnitude of the SCR during fear acquisition was inversely associated with the number of their recorded offences. These findings suggest that juvenile offenders have impairments in the neural systems that subserve emotional learning. The implication is that using punitive measures to control persistent offenders is unlikely to be effective in an identifiable group of juvenile offenders.
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http://dx.doi.org/10.1111/j.1467-7687.2012.01191.xDOI Listing
January 2013

Instance memorization and category influence: challenging the evidence for multiple systems in category learning.

Q J Exp Psychol (Hove) 2013 Jun 12;66(6):1204-26. Epub 2012 Nov 12.

Cardiff University, Cardiff, UK.

A class of dual-system theories of categorization assumes a categorization system based on actively formed prototypes in addition to a separate instance memory system. It has been suggested that, because they have used poorly differentiated category structures (such as the influential "5-4" structure), studies supporting the alternative exemplar theory reveal little about the properties of the categorization system. Dual-system theories assume that the instance memory system only influences categorization behaviour via similarity to single isolated instances, without generalization across instances. However, we present the results of two experiments employing the 5-4 structure to argue against this. Experiment 1 contrasted learning in the standard 5-4 structure with learning in an even more poorly differentiated 5-4 structure. In Experiment 2, participants memorized the 5-4 structure based on a five minute simultaneous presentation of all nine category instances. Both experiments revealed category influences as reflected by differences in instance learnability and generalization, at variance with the dual-system prediction. These results have implications for the exemplars versus prototypes debate and the nature of human categorization mechanisms.
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http://dx.doi.org/10.1080/17470218.2012.735679DOI Listing
June 2013

Affective startle potentiation in juvenile offenders: the role of conduct problems and psychopathic traits.

Soc Neurosci 2013 3;8(2):112-21. Epub 2012 Aug 3.

School of Psychology, Cardiff University, Cardiff, UK.

Emotion processing difficulties are observed in antisocial individuals exhibiting serious antisocial behavior. This study examined emotion processing in 40 male juvenile offenders (JOs) and 52 male controls by measuring startle reflex responses to aversive sounds during the passive viewing of affective and neutral images. JOs as a group exhibited reduced startle-elicited blinks across all slide categories compared to normal controls. Moreover, within the offender group those with more conduct disorder symptoms and higher levels of psychopathic traits displayed reduced startle amplitudes compared to lower-scoring offenders. The finding that startle magnitudes were inversely related to severity of conduct problems supports a dimensional or continuous approach to understanding externalizing disorders. Reductions in amygdala activity could lead to blunted startle magnitudes. The current findings not only provide further evidence that antisocial children have a general defensive motivational system dysfunction and present with impairments in neural systems that subserve emotion processing, but also show for the first time that those with more severe conduct problems have reduced startle responses compared to those who are less severely affected. The implications of these findings for interventions with JOs are discussed.
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http://dx.doi.org/10.1080/17470919.2012.712549DOI Listing
August 2013

Decision making and executive function in male adolescents with early-onset or adolescence-onset conduct disorder and control subjects.

Biol Psychiatry 2009 Jul 10;66(2):162-8. Epub 2009 Apr 10.

Developmental Psychiatry Section, Department of Psychiatry, Cambridge University, Cambridge, United Kingdom.

Background: Although conduct disorder (CD) is associated with an increased susceptibility to substance use disorders, little is known about decision-making processes or reward mechanisms in CD. This study investigated decision making under varying motivational conditions in CD.

Methods: Performances on the Risky Choice Task (RCT) and the Wisconsin Card Sorting Test (WCST) were assessed in 156 adolescents (84 control subjects, 34 with adolescence-onset CD, and 38 with early-onset CD). The RCT was performed twice, once under normal motivational conditions and once under conditions of increased motivation and psychosocial stress.

Results: Increased motivation and stress led to more cautious decision making and changes in framing effects on the RCT in all groups, although such effects were least pronounced in the early-onset CD group. Participants from both CD subgroups selected the risky choice more frequently than control subjects. Under normal motivational conditions, early-onset CD participants chose the risky choice more frequently in trials occurring after small gains, relative to control subjects and adolescence-onset CD participants. Following adjustment for IQ differences, the groups did not differ significantly in terms of WCST performance.

Conclusions: Differences in decision making between control subjects and individuals with CD suggest that the balance between sensitivity to reward and punishment is shifted in this disorder, particularly the early-onset form. Our data on modulation of decision making according to previous outcomes suggest altered reward mechanisms in early-onset CD. The WCST data suggest that impairments in global executive function do not underlie altered decision making in CD.
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http://dx.doi.org/10.1016/j.biopsych.2009.02.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2733860PMC
July 2009

Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

PLoS Genet 2007 May;3(5):e82

Prolexys Pharmaceuticals, Salt Lake City, Utah, United States of America.

Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.
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http://dx.doi.org/10.1371/journal.pgen.0030082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866352PMC
May 2007

Development of an LC-MALDI method for the analysis of protein complexes.

J Am Soc Mass Spectrom 2004 Jun;15(6):803-22

Prolexys Pharmaceuticals, Inc., Salt Lake City, Utah 84116, USA.

In this study, a two-dimensional LC-MALDI-TOF/TOF method has been developed for analyzing protein complexes. In our hands, the method has proven to be an excellent strategy for the analysis of protein complexes isolated in pull-down experiments. This is in part because the preservation of the chromatographic separation on a MALDI target yields an "unlimited" amount of time to obtain MS/MS spectra, making it possible to probe more deeply into complex samples. A brief statistical analysis was performed on the data obtained from the LC-MALDI-TOF/TOF system in order to better understand peptide fragmentation patterns under high-energy collision conditions. These statistical analyses provided some insight into how to evaluate the quality and accuracy of the database search results derived from the TOF/TOF-based analysis. The potential of the method was demonstrated by the successful identification of all the known penicillin-binding proteins in E. coli isolated using a drug-based pull-down with ampicillin as the bait. The performance of the LC-MALDI-TOF/TOF system was compared with that of an equivalent 2D LC-ESI-MS/MS approach, in the analysis of a protein bait-based pull-down. Regardless of the number of peptides identified in the ESI versus MALDI approach, the two approaches were found to be complementary. When the data is merged at the peptide level, the combined result gives higher Mascot scores and an overall higher confidence in protein identification than with either approach alone.
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http://dx.doi.org/10.1016/j.jasms.2004.02.004DOI Listing
June 2004

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins.

Anal Biochem 2003 Jun;317(2):255-8

Myriad Proteomics, Inc., Salt Lake City, UT 84116, USA.

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.
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http://dx.doi.org/10.1016/s0003-2697(03)00116-7DOI Listing
June 2003