Characterization of a Pathogenic DCTN4 Variant

Cytoplasmic cargoes are transported within cells via mechanochemical motor proteins along cytoskeletal filaments such as microtubules. The major minus-end directed, microtubule-based motor, cytoplasmic dynein 1, relies heavily on its binding partner, dynactin, for motor activation and cargo specification. Interactions between dynein and dynactin are scaffolded by cargo-associated adaptor proteins that link the motor complex to diverse cargoes. Dynactin is a large multiprotein complex. Its DCTN4 component has been shown to bind multiple cargo adaptor proteins directly. A genetic variant of DCTN4 (rs35662018, hereafter 018-DCTN4) that yields a tyrosine to cysteine mutation, has been linked to worse disease risk in cystic fibrosis and lung adenocarcinoma. Our group showed previously that expression of the 018-DCTN4 variant impairs cell migration and induces loss of focal adhesions, altered integrin trafficking, and aberrant actin dynamics in a variety of cell lines. The goal of my thesis work was to determine molecular mechanisms underlying the observed phenotypes. My work also provided new insight into the impact of 018-DCTN4 on the function of the lung epithelium. I used a proteomics approach to identify differential interactors that might provide mechanistic explanations for the phenotypes exhibited by cells expressing 018-DCTN4 (Chapter 2). In Chapter 3 I generate a CRISPR gene-edited human bronchial epithelial cell line that expresses 018-DCTN4 from the endogenous locus. Differential transcriptome analysis on these cells indicated changes that explain many of the observed phenotypes. In addition, the transcriptome revealed significant alterations in gene expression patterns relevant to innate immunity and barrier function in the lung. This analysis further revealed decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) and reduction in CFTR protein level. My studies expand our knowledge of the functional impacts of 018-DCTN4 in cells and suggests new mechanistic targets for further exploration

Enantioselective S‐H Insertion Reactions of α‐Carbonyl Sulfoxonium Ylides

The first example of enantioselective S‐H insertion reactions of sulfoxonium ylides is reported. Under the influence of thiourea catalysis, excellent levels of enantiocontrol (up to 95% ee) and yields (up to 97%) are achieved for 31 examples in S‐H insertion reactions of aryl thiols and α‐carbonyl sulfoxonium ylides

Dietary Plant Sterol Esters Must Be Hydrolyzed to Reduce Intestinal Cholesterol Absorption in Hamsters

Background: Elevated concentrations of LDL cholesterol are associated with the development of atherosclerosis and therefore are considered an important target for intervention to prevent cardiovascular diseases. The inhibition of cholesterol absorption in the small intestine is an attractive approach to lowering plasma cholesterol, one that is addressed by drug therapy as well as dietary supplementation with plant sterols and plant sterol esters (PSEs). Objective: This study was conducted to test the hypothesis that the cholesterol-lowering effects of PSE require hydrolysis to free sterols (FSs). Methods: Male Syrian hamsters were fed atherogenic diets (AIN-93M purified diet containing 0.12% cholesterol and 8% coconut oil) to which one of the following was added: no PSEs or ethers (control), 5% sterol stearate esters, 5% sterol palmitate esters (PEs), 5% sterol oleate esters (OEs), 5% sterol stearate ethers (STs; to mimic nonhydrolyzable PSE), or 3% FSs plus 2% sunflower oil. The treatments effectively created a spectrum of PSE hydrolysis across which cholesterol metabolism could be compared. Metabolic measurements included cholesterol absorption, plasma and liver lipid concentration, and fecal neutral sterol and bile acid excretion. Results: The STs and the PEs and SEs were poorly hydrolyzed (1.69–4.12%). In contrast,OEs were 88.3% hydrolyzed. The percent hydrolysis was negatively correlated with cholesterol absorption (r=20.85; P \u3c 0.0001) and positively correlated with fecal cholesterol excretion (r = 0.92; P \u3c 0.0001), suggesting that PSE hydrolysis plays a central role in the cholesterol-lowering properties of PSE. Conclusions: Our data on hamsters suggest that PSE hydrolysis and the presence of FSs is necessary to induce an optimum cholesterol-lowering effect and that poorly hydrolyzed PSEs may lower cholesterol through an alternative mechanism than that of competition with cholesterol for micelle incorporation

Cost-Effectiveness of Male Circumcision for HIV Prevention in a South African Setting

BACKGROUND: Consistent with observational studies, a randomized controlled intervention trial of adult male circumcision (MC) conducted in the general population in Orange Farm (OF) (Gauteng Province, South Africa) demonstrated a protective effect against HIV acquisition of 60%. The objective of this study is to present the first cost-effectiveness analysis of the use of MC as an intervention to reduce the spread of HIV in sub-Saharan Africa. METHODS AND FINDINGS: Cost-effectiveness was modeled for 1,000 MCs done within a general adult male population. Intervention costs included performing MC and treatment of adverse events. HIV prevalence was estimated from published estimates and incidence among susceptible subjects calculated assuming a steady-state epidemic. Effectiveness was defined as the number of HIV infections averted (HIA), which was estimated by dynamically projecting over 20 years the reduction in HIV incidence observed in the OF trial, including secondary transmission to women. Net savings were calculated with adjustment for the averted lifetime duration cost of HIV treatment. Sensitivity analyses examined the effects of input uncertainty and program coverage. All results were discounted to the present at 3% per year. For Gauteng Province, assuming full coverage of the MC intervention, with a 2005 adult male prevalence of 25.6%, 1,000 circumcisions would avert an estimated 308 (80% CI 189–428) infections over 20 years. The cost is $181 (80$117–$306) per HIA, and net savings are$2.4 million (80% CI $1.3 million to$3.6 million). Cost-effectiveness is sensitive to the costs of MC and of averted HIV treatment, the protective effect of MC, and HIV prevalence. With an HIV prevalence of 8.4%, the cost per HIA is $551 (80$344–$1,071) and net savings are$753,000 (80% CI $0.3 million to$1.2 million). Cost-effectiveness improves by less than 10% when MC intervention coverage is 50% of full coverage. CONCLUSIONS: In settings in sub-Saharan Africa with high or moderate HIV prevalence among the general population, adult MC is likely to be a cost-effective HIV prevention strategy, even when it has a low coverage. MC generates large net savings after adjustment for averted HIV medical costs

Grant Cycles, Deadlines, and Labor Advocacy: The Changing Work of Project Archivists

Presentation from the MARAC conference in Pittsburgh, PA on April 14–16, 2016. S7 - Grant Cycles, Deadlines, and Labor Advocacy: The Changing Work of Project Archivists

Nicotine abolishes the regulatory function of rapamycin on memory programming.

<p>Sorted OT-I cells were stimulated for 3 days with 3SI in the presence or absence of nicotine at 10 µM. Rapamycin was used in combination with or without nicotine. Cells were harvested and transferred into recipient B6 mice at a concentration of 10⁶ cells/mouse. Memory CTLs were examined 40 days post-transfer. A. Comparison of memory CTLs in spleen. B. Comparison of the phenotype of memory CTLs in spleen. C. Comparison of memory protection against LM-OVA challenge in different memory mice in Panel A. D. Secondary expansion of memory CTLs 3 days post-LM-OVA challenge. These animals were the same as mentioned in Panel C. The results are representatives of two separate experiments with similar results. Asterisks indicate statistical significance. *, P<0.05; **, P<0.01; ***, P<0.001. E. Sorted OT-I cells were stimulated for 3 days with 3SI in the presence or absence of rapamycin, and expression of nAChRs was compared relatively to β1 in 3SI stimulation using quantitative PCR. Dotted lines in A and C indicate the detection limits. The results are representatives of two separate experiments with similar results.</p

Nicotine Inhibits Memory CTL Programming

<div><p>Nicotine is the main tobacco component responsible for tobacco addiction and is used extensively in smoking and smoking cessation therapies. However, little is known about its effects on the immune system. We confirmed that multiple nicotinic receptors are expressed on mouse and human cytotoxic T lymphocytes (CTLs) and demonstrated that nicotinic receptors on mouse CTLs are regulated during activation. Acute nicotine presence during activation increases primary CTL expansion <i>in vitro</i>, but impairs <i>in vivo</i> expansion after transfer and subsequent memory CTL differentiation, which reduces protection against subsequent pathogen challenges. Furthermore, nicotine abolishes the regulatory effect of rapamycin on memory CTL programming, which can be attributed to the fact that rapamycin enhances expression of nicotinic receptors. Interestingly, naïve CTLs from chronic nicotine-treated mice have normal memory programming, which is impaired by nicotine during activation <i>in vitro</i>. In conclusion, simultaneous exposure to nicotine and antigen during CTL activation negatively affects memory development.</p></div

Chronic exposure to nicotine does not change naïve CTL’s memory programming.

<p>OT-I transgenic mice were given drinking water supplemented with nicotine at 200 µg/ml for 60 days <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Qian1" target="_blank">[34]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Lawson1" target="_blank">[37]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Wang1" target="_blank">[39]</a>. Naïve OT-I cells were purified from the nicotine treated OT-I mice or un-treated control mice and stimulated for 3 days with 3SI in the presence or absence of nicotine at 10 µM. Rapamycin was used with or without nicotine. Cells were then harvested for adoptive transfer at a concentration of 10⁶ cells/mouse for memory differentiation in recipient B6 mice <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Xiao1" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Rao1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068183#pone.0068183-Li2" target="_blank">[25]</a> (A–E) or direct examination (F–G). A–C. Analysis of memory CTLs 30 days after transfer. A. Comparison of memory CTLs in blood programmed <i>in vitro</i> by IL-12 from control or nicotine experienced OT-I mice. B. Comparison of memory CTLs in blood programmed under different treatments. All of the naïve OT-I CTLs were from nicotine treated donor mice. Data for “NC water experienced” in (A) are the same as “Control” in (B). C. CD62L expression on memory CTLs from blood in (B). D. Memory mice from (B) were challenged with LM-OVA, and protection was compared 3 days after challenge in spleen. E. Memory CTL secondary expansion in the blood 3 days after LM-OVA challenged (the same mice in D) mice. F–G. <i>In vitro</i> stimulated cells (for 3 days) were harvested to examine the production of functional molecules (F) or transcription factors and mTOR signaling molecules (G). Data from one experiment is shown, representative of two other experiments with similar results. Asterisks indicate statistical significance. *, P<0.05; **, P<0.01; ***, P<0.001, NS, not significant.</p