Characterization of the role of BGS13 in the secretory mechanism of Pichia pastoris

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker’s yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain’s cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits

Nintedanib for Systemic Sclerosis-Associated Interstitial Lung Disease

BACKGROUND: Interstitial lung disease (ILD) is a common manifestation of systemic sclerosis and a leading cause of systemic sclerosis-related death. Nintedanib, a tyrosine kinase inhibitor, has been shown to have antifibrotic and antiinflammatory effects in preclinical models of systemic sclerosis and ILD. METHODS: We conducted a randomized, double-blind, placebo-controlled trial to investigate the efficacy and safety of nintedanib in patients with ILD associated with systemic sclerosis. Patients who had systemic sclerosis with an onset of the first non-Raynaud's symptom within the past 7 years and a high-resolution computed tomographic scan that showed fibrosis affecting at least 10% of the lungs were randomly assigned, in a 1:1 ratio, to receive 150 mg of nintedanib, administered orally twice daily, or placebo. The primary end point was the annual rate of decline in forced vital capacity (FVC), assessed over a 52-week period. Key secondary end points were absolute changes from baseline in the modified Rodnan skin score and in the total score on the St. George's Respiratory Questionnaire (SGRQ) at week 52. RESULTS: A total of 576 patients received at least one dose of nintedanib or placebo; 51.9% had diffuse cutaneous systemic sclerosis, and 48.4% were receiving mycophenolate at baseline. In the primary end-point analysis, the adjusted annual rate of change in FVC was 1252.4 ml per year in the nintedanib group and 1293.3 ml per year in the placebo group (difference, 41.0 ml per year; 95% confidence interval [CI], 2.9 to 79.0; P=0.04). Sensitivity analyses based on multiple imputation for missing data yielded P values for the primary end point ranging from 0.06 to 0.10. The change from baseline in the modified Rodnan skin score and the total score on the SGRQ at week 52 did not differ significantly between the trial groups, with differences of 120.21 (95% CI, 120.94 to 0.53; P=0.58) and 1.69 (95% CI, 120.73 to 4.12 [not adjusted for multiple comparisons]), respectively. Diarrhea, the most common adverse event, was reported in 75.7% of the patients in the nintedanib group and in 31.6% of those in the placebo group. CONCLUSIONS: Among patients with ILD associated with systemic sclerosis, the annual rate of decline in FVC was lower with nintedanib than with placebo; no clinical benefit of nintedanib was observed for other manifestations of systemic sclerosis. The adverse-event profile of nintedanib observed in this trial was similar to that observed in patients with idiopathic pulmonary fibrosis; gastrointestinal adverse events, including diarrhea, were more common with nintedanib than with placebo

Fake News/News Literacy

A presentation on how to become media literate.FAKE NEWS/NEWS LITERACY CHRISTINE MOUA – LIBRARY ASSOCIATE STEPHEN D. MILLER – ASSOCIATE VICE PROVOST, UMUC LIBRARY MARCH 1, 2017WHAT IS FAKE NEWS Fake News Clickbait Why is this a library related issue? Silverman, C. (2016). Coverly, D. (2014). Filter the feed. (2016).LET’S MAKE FAKE NEWS! Jacks Films, Fake News, (2017). RECENT “FAKE NEWS” ALLEGATIONS Getty Images. (2017). EROSION OF TRUST Pew Research Center • 1958 – when first asked question about trust between civilians and Govt – 73% • Dropped tremendously, American’s trust in government – 19% Pew Research Center, 2015, Trust in GovernmentTRUST IN MASS MEDIA DECLINING (Swift, A., 2016, September 14)Edelman (2017) - http://www.edelman.com/trust2017/WHAT IS NEWS LITERACY? International Federation of Library Association. (2017). How to spot fake news [infographic]. PURPOSE OF NEWS: Alert us Connect us Divert us Salvator, R. & Kattalia, K. (2015) TRADITIONAL NEWS SOURCES Main Stream news sources Social Media News Entertainment news (Votero, V. (2017). News Quality Chart. NEWS/FILTER BUBBLES How do you make sure reports/news is fair? • Fair play • Fair language • Fair presentation (Digital Resource Center, 2015, News Literacy Basics) EVALUATE SOURCES/ TIPS How to evaluate sources • VIA – Verify, Independence, Accountability (Digital Resource Center, 2015, News Literacy Basics) • IMVA/IN – Independent, Multiple sources, Verify, Authoritative/Informed, Named Sources (Digital Resource Center, 2015, News Literacy Basics) • C.R.A.A.P. – Currency, relevance, authority, accuracy, purpose (Wichowski & Laura, 2013) American Library Association, (2015 HELPFUL RESOURCES: UMUC Library – Fake news guide (http://sites.umuc.edu/library/libhow/credibility.cfm#fakenews) Pratt Library (http://www.prattlibrary.org/research/tools/index.aspx?cat=19941&id=85681) NY Magazine - Helpful list of fake and misleading sites (http://nymag.com/selectall/2016/11/fake-facebook-news-sites-to-avoid.html) • False, misleading, clickbait, or satirical news sites (https://docs.google.com/document/d/10eA5-mCZLSS4MQY5QGb5ewC3VAL6pLkT53V_81ZyitM/preview) Snopes (www.snopes.com) Politifact (http://www.politifact.com/) Fact Check (http://www.factcheck.org/) Society of Professional Journalists (http://www.spj.org/ethicscode.asp) Contact a librarian! We are always here to help! [email protected] IMAGE CREDITS/CITATIONS: American Library Association, (2015). Libraries Transform Toolkit. Retrieved from http://ilovelibraries.org/librariestransform/toolkit Coverly, D. (2014). Clickbait [cartoon]. Retrieved from http://www.speedbump.com/index.html Department of State. (2017). U.S. Diplomacy and Yellow Journalism, 1895 -1898. Retrieved from https://history.state.gov/milestones/1866-1898/yellow-journalism.) Edelman (2017). 2017 Edelman trust barometer. http://www.edelman.com/trust2017/ Filter the feed. (2016). Hillary Clinton adopts alien baby [image]. Retrieved from https://filterthefeed.files.wordpress.com/2016/12/hvb.jpg Getty Images. (2017). Trump VS. the media [image]. Retrieved from http://i2.cdn.turner.com/money/dam/assets/151207141010-donald-trump-vs-the-media-00000614-1280x720.jpg Google. (2017). [whisper image]. Retrieved from https://www.google.com/images Google. (2017). [Social media images]. Retrieved from https://www.google.com/images International Federation of Library Associations. (2017). How to spot fake news [infographic]. Retrieved from http://www.ifla.org/publications/node/11174 Jack’s Films. (2017, February). Fake news: let’s fool the world! (YIAY #314) [Youtube video]. Retrieved from https://www.youtube.com/watch?v=2PcwpLRF-Go People magazine. (2017). [February 2017 cover]. Retrieved from http://www.people.com Pew Research Center (2015, November 23). Trust in government: 1958-2015. Retrieved from http://www.people-press.org/2015/11/23/1-trust-in-government-1958-2015/ Salvator, R. & Kattalia, K. (2015). Hurricane Sandy four years later: Remembering the historic storm and the aftermath [American flag]. Retrieved from http://www.nydailynews.com/news/hurricane-sandy-strikes-east-coast-gallery-1.1194577?pmSlide=1.1991236 Silverman, C. (2016). Here are 50 of the biggest fake news hits on Facebook in 2016 [top 5 list]. Retrieved from http://www.buzzfeed.com Stony Brook Center for News Literacy. (2014). News Literacy Basics. Retrieved from http://www. http://drc.centerfornewsliteracy.org/content/news-literacy-basics Swift, A. (2016, September 14). Americans’ trust in mass media sinks to new low. Retrieved from Gallup, http://www.gallup.com/poll/195542/americans-trust-mass-media-sinks-new-low.aspx Votero, V. (2017). News Quality Chart. Retrieved from http://www.allgeneralizationsarefalse.com/?p=53 Wichowski, D.E. & Laura, E.K. (2013). Establishing Credibility in the information jungle: blogs, microblogs, and the CRAAP test. Library Staff Journal Articles, 229-251. http://dx.doi.org/10.4018/978-1-4666-2663-8.ch01

Core Structure and Surface Functionalization of Carbon Nanomaterials Alter Impacts to Daphnid Mortality, Reproduction, and Growth: Acute Assays Do Not Predict Chronic Exposure Impacts

There are currently over ninety products incorporating carbon nanomaterials (CNMs) on the market today for a variety of applications. Modifications in core structure and surface chemistry of manufactured nanomaterials are used to optimize nanomaterials for specific uses. However, there is a notable lack of information on how core structure and surface chemistry may alter toxicity in low-level, chronic exposures. This paper examines the effects of twelve CNMs that differ in their core structure and surface chemistry to <i>Daphnia magna</i> over a 21-day chronic exposure. Overall, nanomaterials with a carbon nanotube core were more toxic to daphnids than fullerenes, with the one exception of fullerenes with a gamma-cyclodextrin surface chemistry. Acute mortality was not a good predictor of chronic effects as none of the CNMs induced toxicity at tested concentrations after 48 h, yet chronic assays indicated significant differences in mortality, reproduction, and growth realized after 21 days. Our results indicate that (1) acute exposure assays do not accurately describe the impact of CNMs to biological systems, (2) chronic exposures provide valuable information that indicates the potential for different modes of action for nanomaterials of differing chemistries, and (3) core structure and surface chemistry both influence particle toxicity

Propriétés virologiques et sérologiques de l’infection à virus Chikungunya : bilan de l’épidémie dans les DFA

International audienc

Junctional trafficking and restoration of retrograde signaling by the cytoplasmic RyR1 domain

The type 1 ryanodine receptor (RyR1) in skeletal muscle is a homotetrameric protein that releases Ca2+ from the sarcoplasmic reticulum (SR) in response to an orthograde signal from the dihydropyridine receptor (DHPR) in the plasma membrane (PM). Additionally, a retrograde signal from RyR1 increases the amplitude of the Ca2+ current produced by Ca(v)1.1, the principle subunit of the DHPR. This bidirectional signaling is thought to depend on physical links, of unknown identity, between the DHPR and RyR1. Here, we investigate whether the isolated cytoplasmic domain of RyR1 can interact structurally or functionally with Ca(v)1.1 by producing an N-terminal construct (RyR1(1)(:)(4300)) that lacks the C-terminal membrane domain. In Ca(v)1.1-null (dysgenic) myotubes, RyR1(1)(:)(4300) is diffusely distributed, but in RyR1-null (dyspedic) myotubes it localizes in puncta at SR-PM junctions containing endogenous Ca(v)1.1. Fluorescence recovery after photobleaching indicates that diffuse RyR1(1)(:)(4300) is mobile, whereas resistance to being washed out with a large-bore micropipette indicates that the punctate RyR1(1)(:)(4300) stably associates with PM-SR junctions. Strikingly, expression of RyR1(1)(:)(4300) in dyspedic myotubes causes an increased amplitude, and slowed activation, of Ca2+ current through Ca(v)1.1, which is almost identical to the effects of full-length RyR1. Fast protein liquid chromatography indicates that similar to 25% of RyR1(1)(:)(4300) in diluted cytosolic lysate of transfected tsA201 cells is present in complexes larger in size than the monomer, and intermolecular fluorescence resonance energy transfer implies that RyR1(1)(:)(4300) is significantly oligomerized within intact tsA201 cells and dyspedic myotubes. A large fraction of these oligomers may be homotetramers because freeze-fracture electron micrographs reveal that the frequency of particles arranged like DH PR tetrads is substantially increased by transfecting RyR-null myotubes with RyR1(1)(:)(4300) . In summary, the RyR1 cytoplasmic domain, separated from its SR membrane anchor, retains a tendency toward oligomerization/tetramerization, binds to SR-PM junctions in myotubes only if Ca(v)1.1 is also present and is fully functional in retrograde signaling to Ca(v)1.1

Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris

The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris

The use of pretest probability increases the value of high-resolution CT in diagnosing usual interstitial pneumonia

BackgroundRecent studies have suggested that non-definitive patterns on high-resolution CT (HRCT) scan provide sufficient diagnostic specificity to forgo surgical lung biopsy in the diagnosis of idiopathic pulmonary fibrosis (IPF). The objective of this study was to determine test characteristics of non-definitive HRCT patterns for identifying histopathological usual interstitial pneumonia (UIP).MethodsPatients with biopsy-proven interstitial lung disease (ILD) and non-definitive HRCT scans were identified from two academic ILD centres. Test characteristics for HRCT patterns as predictors of UIP on surgical lung biopsy were derived and validated in independent cohorts.ResultsIn the derivation cohort, 64/385 (17%) had possible UIP pattern on HRCT; 321/385 (83%) had inconsistent with UIP pattern. 113/385 (29%) patients had histopathological UIP pattern in the derivation cohort. Possible UIP pattern had a specificity of 91.2% (95% CI 87.2% to 94.3%) and a positive predictive value (PPV) of 62.5% (95% CI 49.5% to 74.3%) for UIP pattern on surgical lung biopsy. The addition of age, sex and total traction bronchiectasis score improved the PPV. Inconsistent with UIP pattern demonstrated poor PPV (22.7%, 95% CI 18.3% to 27.7%). HRCT pattern specificity was nearly identical in the validation cohort (92.7%, 95% CI 82.4% to 98.0%). The substantially higher prevalence of UIP pattern in the validation cohort improved the PPV of HRCT patterns.ConclusionsA possible UIP pattern on HRCT has high specificity for UIP on surgical lung biopsy, but PPV is highly dependent on underlying prevalence. Adding clinical and radiographic features to possible UIP pattern on HRCT may provide sufficient probability of histopathological UIP across prevalence ranges to change clinical decision-making