Faculty Bibliography

BACKGROUND: Measurement of the global burden of disease with disability-adjusted life-years (DALYs) requires disability weights that quantify health losses for all non-fatal consequences of disease and injury. There has been extensive debate about a range of conceptual and methodological issues concerning the definition and measurement of these weights. Our primary objective was a comprehensive re-estimation of disability weights for the Global Burden of Disease Study 2010 through a large-scale empirical investigation in which judgments about health losses associated with many causes of disease and injury were elicited from the general public in diverse communities through a new, standardised approach. METHODS: We surveyed respondents in two ways: household surveys of adults aged 18 years or older (face-to-face interviews in Bangladesh, Indonesia, Peru, and Tanzania; telephone interviews in the USA) between Oct 28, 2009, and June 23, 2010; and an open-access web-based survey between July 26, 2010, and May 16, 2011. The surveys used paired comparison questions, in which respondents considered two hypothetical individuals with different, randomly selected health states and indicated which person they regarded as healthier. The web survey added questions about population health equivalence, which compared the overall health benefits of different life-saving or disease-prevention programmes. We analysed paired comparison responses with probit regression analysis on all 220 unique states in the study. We used results from the population health equivalence responses to anchor the results from the paired comparisons on the disability weight scale from 0 (implying no loss of health) to 1 (implying a health loss equivalent to death). Additionally, we compared new disability weights with those used in WHO's most recent update of the Global Burden of Disease Study for 2004. FINDINGS: 13,902 individuals participated in household surveys and 16,328 in the web survey. Analysis of paired comparison responses indicated a high degree of consistency across surveys: correlations between individual survey results and results from analysis of the pooled dataset were 0.9 or higher in all surveys except in Bangladesh (r=0.75). Most of the 220 disability weights were located on the mild end of the severity scale, with 58 (26%) having weights below 0.05. Five (11%) states had weights below 0.01, such as mild anaemia, mild hearing or vision loss, and secondary infertility. The health states with the highest disability weights were acute schizophrenia (0.76) and severe multiple sclerosis (0.71). We identified a broad pattern of agreement between the old and new weights (r=0.70), particularly in the moderate-to-severe range. However, in the mild range below 0.2, many states had significantly lower weights in our study than previously. INTERPRETATION: This study represents the most extensive empirical effort as yet to measure disability weights. By contrast with the popular hypothesis that disability assessments vary widely across samples with different cultural environments, we have reported strong evidence of highly consistent results. FUNDING: Bill & Melinda Gates Foundation.

Fusion of lysosomes with the plasma membrane is a calcium-dependent process that is crucial for membrane repair, limiting pathogen entry and clearing cellular debris. In non-polarized cells, lysosome exocytosis facilitates rapid resealing of torn membranes. Here, we investigate the mechanism of lysosome exocytosis in polarized epithelia, the main barrier between the organism and the external environment and the first line of defense against pathogens. We find that in polarized Madin-Darby canine kidney (MDCK) cells, calcium ionophores or pore-forming toxins cause lysosomes to fuse predominantly with the basolateral membrane. This polarized exocytosis is regulated by the actin cytoskeleton, membrane cholesterol and the clathrin adaptor AP-1. Depolymerization of actin, but not microtubules, causes apical lysosome fusion, supporting the hypothesis that cortical actin is a barrier to exocytosis. Overloading lysosomes with cholesterol inhibits exocytosis, suggesting that excess cholesterol paralyzes lysosomal traffic. The clathrin adaptor AP-1 is responsible for accurately targeting syntaxin 4 to the basolateral domain. In cells lacking either the ubiquitous AP-1A or the epithelial-specific AP-1B, syntaxin 4 is non-polar. This causes lysosomes to fuse with both the apical and basolateral membranes. Consistent with these findings, RNAi-mediated depletion of syntaxin 4 inhibits basolateral exocytosis in wild-type MDCK, and both apical and basolateral exocytosis in cells lacking AP-1A or AP-1B. Our results provide fundamental insight into the molecular machinery involved in membrane repair in polarized epithelia and suggest that AP-1 is a crucial regulator of this process.

Both metastatic cancer cells and immune cells pursuing infections must penetrate the endothelial barrier presented by the blood vessel wall to reach tissues. This transmigration requires the Rap1 GTPase to modulate Integrin affinity. We have defined a new model system in Drosophila to dissect the genetic pathways that control this process. Using live imaging and genetics, we have discovered that Drosophila melanogaster immune cells penetrate an epithelial, DE-Cadherin-based tissue barrier during their embryonic developmental movements into the tail. A mutant in RhoL, a GTPase homolog that is only expressed in Drosophila macrophages at this stage, specifically blocks this invasive step but not other aspects of guided migration. The invasion defect in rhoL mutants can be rescued by lowering embryonic DECadherin levels, arguing that RhoL is required to allow hemocytes to penetrate adherens junctions. We also show that RhoL is localized to the cell surface and mediates Integrin adhesion in hemocytes. We observe that RhoL mutants fail to transport the Integrin activator Rap1 from a cytoplasmic membrane containing concentration to the leading edge during invasion. We have identified 4 mutants in secretory pathway components with defects in this invasive migration, one of which has been linked to metastasis in mice. These genes are expressed specifically during the invasive phase and turn off thereafter. Rap1 localization is found to be dynamically cycling to the cell surface at this time. However after this, Rap1 is found mostly in an intracellular concentration even in wild type. We have thus identified RhoL as a new regulator of invasion, adhesion and Rap1 localization. We propose that RhoL is part of a regulated intracellular trafficking pathway required to shuttle the Rap1 Integrin activator to and from the cytoplasmic edge, permitting the dynamic shifts in adhesion required for hemocytes to invade between adherens junctions. This capacity for invasion and Rap1 relocalization appears to be develo!

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by the production of antinuclear autoantibodies. Antinuclear autoantibody development is recognized as one of the initial stages of disease that often results in systemic inflammation, kidney disease, and death. The etiology is complex, but it is clear that innate pathways may play an important role in disease progression. Recent data have highlighted an important role for the TLR family, particularly TLR7, in both human disease and murine models. In this study, we have presented a low copy conditional TLR7 transgenic (Tg7) mouse strain that does not develop spontaneous autoimmunity. When we combine Tg7 with the Sle1 lupus susceptibility locus, the mice develop severe disease. Using the CD19(Cre) recombinase system, we normalized expression of TLR7 solely within the B cells. Using this method we demonstrated that overexpression of TLR7 within the B cell compartment reduces the marginal zone B cell compartment and increases B and T cell activation but not T follicular helper cell development. Moreover, this enhanced B cell TLR7 expression permits the specific development of Abs to RNA/protein complexes and exacerbates SLE disease.

Co-amplification of transgenes using the dihydrofolate reductase/methotrexate (DHFR/MTX) system is a widely used method for the isolation of Chinese hamster ovary (CHO) cell lines that secrete high levels of recombinant proteins. A bottleneck in this process is the stepwise selection for MTX resistant populations; which can be slow, tedious and erratic. We sought to speed up and regularize this process by isolating dhfr(-) CHO cell lines capable of integrating a transgene of interest into a defined chromosomal location that supports a high rate of gene amplification. We isolated 100 independent transfectants carrying a gene for human adenosine deaminase (ada) linked to a phiC31 attP site and a portion of the dihydrofolate reductase (dhfr) gene. Measurement of the ada amplification rate in each transfectant using Luria-Delbruck fluctuation analysis revealed a wide clonal variation; sub-cloning showed these rates to be heritable. Site directed recombination was used to insert a transgene carrying a reporter gene for secreted embryonic alkaline phosphatase (SEAP) as well as the remainder of the dhfr gene into the attP site at this location in several of these clones. Subsequent selection for gene amplification of the reconstructed dhfr gene in a high ada amplification candidate clone (DG44-HA-4) yielded reproducible rates of seap gene amplification and concomitant increased levels of SEAP secretion. In contrast, random integrations of the dhfr gene into clone HA-4 did not yield these high levels of amplification. This cell line as well as this method of screening for high amplification rates may prove helpful for the reliable amplification of recombinant genes for therapeutically or diagnostically useful proteins.

Voltage-gated K(+) channels underlie the electrical excitability of cells. Each subunit of the functional tetramer consists of the tandem fusion of two modules, an N-terminal voltage-sensor and a C-terminal pore. To investigate how sensor coupling to the pore generates voltage-dependent channel opening, we solved the crystal structure and characterized the function of a voltage-gated K(+) channel pore in a lipid membrane. The structure of a functional channel in a membrane environment at 3.1 A resolution establishes an unprecedented connection between channel structure and function. The structure is unique in delineating an ion-occupied ready to conduct selectivity filter, a confined aqueous cavity, and a closed activation gate, embodying a dynamic entity trapped in an unstable closed state.

Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2-releasing poly-L-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of NQO1 gene in mice leads to increased susceptibility to myeloproliferative disease. In this report, we demonstrate that NQO1 controls the stability of myeloid differentiation factor C/EBPalpha against 20S proteasomal degradation during radiation exposure stress. Co-immunoprecipitation studies showed that NQO1, C/EBPalpha, and 20S all interacted with each other. C/EBPalpha interaction with 20S led to the degradation of C/EBPalpha. NQO1 in presence of its cofactor NADH protected C/EBPalpha against 20S degradation. Deletion and site-directed mutagenesis demonstrated that NQO1 and 20S competed for the same binding region (268)SGAGAGKAKKSV(279) in C/EBPalpha. Mutagenesis studies also revealed that NQO1Y127/Y129 required for NADH binding is essential for NQO1 stabilization of C/EBPalpha. Exposure of mice and HL-60 cells to 3 Grays of gamma-radiation led to increased NQO1 that stabilized C/EBPalpha against 20S proteasomal degradation. This mechanism of NQO1 regulation of C/EBPalpha may provide protection to bone marrow against adverse effects of radiation exposure. The studies have significance for human individuals carrying hetero- or homozygous NQO1P187S mutation and are deficient or lack NQO1 protein.

Although transcriptional regulation of stem cell pluripotency and differentiation has been extensively studied, only a small number of studies have addressed the roles for posttranslational modifications in these processes. A key mechanism of posttranslational modification is ubiquitination by the ubiquitin-proteasome system (UPS). Here, using shotgun proteomics, we map the ubiquitinated protein landscape during embryonic stem cell (ESC) differentiation and induced pluripotency. Moreover, using UPS-targeted RNAi screens, we identify additional regulators of pluripotency and differentiation. We focus on two of these proteins, the deubiquitinating enzyme Psmd14 and the E3 ligase Fbxw7, and characterize their importance in ESC pluripotency and cellular reprogramming. This global characterization of the UPS as a key regulator of stem cell pluripotency opens the way for future studies that focus on specific UPS enzymes or ubiquitinated substrates.

BACKGROUND: Companion diagnostic tests can depend on accurate measurement of protein expression in tissues. Preanalytic variables, especially cold ischemic time (time from tissue removal to fixation in formalin) can affect the measurement and may cause false-negative results. We examined 23 proteins, including four commonly used breast cancer biomarker proteins, to quantify their sensitivity to cold ischemia in breast cancer tissues. METHODS: A series of 93 breast cancer specimens with known time-to-fixation represented in a tissue microarray and a second series of 25 matched pairs of core needle biopsies and breast cancer resections were used to evaluate changes in antigenicity as a function of cold ischemic time. Estrogen receptor (ER), progesterone receptor (PgR), HER2 or Ki67, and 19 other antigens were tested. Each antigen was measured using the AQUA method of quantitative immunofluorescence on at least one series. All statistical tests were two-sided. RESULTS: We found no evidence for loss of antigenicity with time-to-fixation for ER, PgR, HER2, or Ki67 in a 4-hour time window. However, with a bootstrapping analysis, we observed a trend toward loss for ER and PgR, a statistically significant loss of antigenicity for phosphorylated tyrosine (P = .0048), and trends toward loss for other proteins. There was evidence of increased antigenicity in acetylated lysine, AKAP13 (P = .009), and HIF1A (P = .046), which are proteins known to be expressed in conditions of hypoxia. The loss of antigenicity for phosphorylated tyrosine and increase in expression of AKAP13, and HIF1A were confirmed in the biopsy/resection series. CONCLUSIONS: Key breast cancer biomarkers show no evidence of loss of antigenicity, although this dataset assesses the relatively short time beyond the 1-hour limit in recent guidelines. Other proteins show changes in antigenicity in both directions. Future studies that extend the time range and normalize for heterogeneity will provide more comprehensive information on preanalytic variation due to cold ischemic time.