Faculty Bibliography
Searched for:
school:SOM
Department/Unit:Cell Biology
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14017
Mechanism of polarized lysosome exocytosis in epithelial cells
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.
PMCID:3585513
PMID: 23038769
ISSN: 0021-9533
CID: 375082
Rap1 localization is dynamically controlled during immune cell transmigration in Drosophila [Meeting Abstract]
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!
EMBASE:71413647
ISSN: 1059-1524
CID: 884452
A comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010
BACKGROUND: Quantification of the disease burden caused by different risks informs prevention by providing an account of health loss different to that provided by a disease-by-disease analysis. No complete revision of global disease burden caused by risk factors has been done since a comparative risk assessment in 2000, and no previous analysis has assessed changes in burden attributable to risk factors over time. METHODS: We estimated deaths and disability-adjusted life years (DALYs; sum of years lived with disability [YLD] and years of life lost [YLL]) attributable to the independent effects of 67 risk factors and clusters of risk factors for 21 regions in 1990 and 2010. We estimated exposure distributions for each year, region, sex, and age group, and relative risks per unit of exposure by systematically reviewing and synthesising published and unpublished data. We used these estimates, together with estimates of cause-specific deaths and DALYs from the Global Burden of Disease Study 2010, to calculate the burden attributable to each risk factor exposure compared with the theoretical-minimum-risk exposure. We incorporated uncertainty in disease burden, relative risks, and exposures into our estimates of attributable burden. FINDINGS: In 2010, the three leading risk factors for global disease burden were high blood pressure (7.0% [95% uncertainty interval 6.2-7.7] of global DALYs), tobacco smoking including second-hand smoke (6.3% [5.5-7.0]), and alcohol use (5.5% [5.0-5.9]). In 1990, the leading risks were childhood underweight (7.9% [6.8-9.4]), household air pollution from solid fuels (HAP; 7.0% [5.6-8.3]), and tobacco smoking including second-hand smoke (6.1% [5.4-6.8]). Dietary risk factors and physical inactivity collectively accounted for 10.0% (95% UI 9.2-10.8) of global DALYs in 2010, with the most prominent dietary risks being diets low in fruits and those high in sodium. Several risks that primarily affect childhood communicable diseases, including unimproved water and sanitation and childhood micronutrient deficiencies, fell in rank between 1990 and 2010, with unimproved water and sanitation accounting for 0.9% (0.4-1.6) of global DALYs in 2010. However, in most of sub-Saharan Africa childhood underweight, HAP, and non-exclusive and discontinued breastfeeding were the leading risks in 2010, while HAP was the leading risk in south Asia. The leading risk factor in Eastern Europe, most of Latin America, and southern sub-Saharan Africa in 2010 was alcohol use; in most of Asia, North Africa and Middle East, and central Europe it was high blood pressure. Despite declines, tobacco smoking including second-hand smoke remained the leading risk in high-income north America and western Europe. High body-mass index has increased globally and it is the leading risk in Australasia and southern Latin America, and also ranks high in other high-income regions, North Africa and Middle East, and Oceania. INTERPRETATION: Worldwide, the contribution of different risk factors to disease burden has changed substantially, with a shift away from risks for communicable diseases in children towards those for non-communicable diseases in adults. These changes are related to the ageing population, decreased mortality among children younger than 5 years, changes in cause-of-death composition, and changes in risk factor exposures. New evidence has led to changes in the magnitude of key risks including unimproved water and sanitation, vitamin A and zinc deficiencies, and ambient particulate matter pollution. The extent to which the epidemiological shift has occurred and what the leading risks currently are varies greatly across regions. In much of sub-Saharan Africa, the leading risks are still those associated with poverty and those that affect children. FUNDING: Bill & Melinda Gates Foundation.
PMCID:4156511
PMID: 23245609
ISSN: 0140-6736
CID: 209482
Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010
BACKGROUND: Non-fatal health outcomes from diseases and injuries are a crucial consideration in the promotion and monitoring of individual and population health. The Global Burden of Disease (GBD) studies done in 1990 and 2000 have been the only studies to quantify non-fatal health outcomes across an exhaustive set of disorders at the global and regional level. Neither effort quantified uncertainty in prevalence or years lived with disability (YLDs). METHODS: Of the 291 diseases and injuries in the GBD cause list, 289 cause disability. For 1160 sequelae of the 289 diseases and injuries, we undertook a systematic analysis of prevalence, incidence, remission, duration, and excess mortality. Sources included published studies, case notification, population-based cancer registries, other disease registries, antenatal clinic serosurveillance, hospital discharge data, ambulatory care data, household surveys, other surveys, and cohort studies. For most sequelae, we used a Bayesian meta-regression method, DisMod-MR, designed to address key limitations in descriptive epidemiological data, including missing data, inconsistency, and large methodological variation between data sources. For some disorders, we used natural history models, geospatial models, back-calculation models (models calculating incidence from population mortality rates and case fatality), or registration completeness models (models adjusting for incomplete registration with health-system access and other covariates). Disability weights for 220 unique health states were used to capture the severity of health loss. YLDs by cause at age, sex, country, and year levels were adjusted for comorbidity with simulation methods. We included uncertainty estimates at all stages of the analysis. FINDINGS: Global prevalence for all ages combined in 2010 across the 1160 sequelae ranged from fewer than one case per 1 million people to 350,000 cases per 1 million people. Prevalence and severity of health loss were weakly correlated (correlation coefficient -0.37). In 2010, there were 777 million YLDs from all causes, up from 583 million in 1990. The main contributors to global YLDs were mental and behavioural disorders, musculoskeletal disorders, and diabetes or endocrine diseases. The leading specific causes of YLDs were much the same in 2010 as they were in 1990: low back pain, major depressive disorder, iron-deficiency anaemia, neck pain, chronic obstructive pulmonary disease, anxiety disorders, migraine, diabetes, and falls. Age-specific prevalence of YLDs increased with age in all regions and has decreased slightly from 1990 to 2010. Regional patterns of the leading causes of YLDs were more similar compared with years of life lost due to premature mortality. Neglected tropical diseases, HIV/AIDS, tuberculosis, malaria, and anaemia were important causes of YLDs in sub-Saharan Africa. INTERPRETATION: Rates of YLDs per 100,000 people have remained largely constant over time but rise steadily with age. Population growth and ageing have increased YLD numbers and crude rates over the past two decades. Prevalences of the most common causes of YLDs, such as mental and behavioural disorders and musculoskeletal disorders, have not decreased. Health systems will need to address the needs of the rising numbers of individuals with a range of disorders that largely cause disability but not mortality. Quantification of the burden of non-fatal health outcomes will be crucial to understand how well health systems are responding to these challenges. Effective and affordable strategies to deal with this rising burden are an urgent priority for health systems in most parts of the world. FUNDING: Bill & Melinda Gates Foundation.
PMID: 23245607
ISSN: 0140-6736
CID: 209492
Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990-2010: a systematic analysis for the Global Burden of Disease Study 2010
BACKGROUND: Measuring disease and injury burden in populations requires a composite metric that captures both premature mortality and the prevalence and severity of ill-health. The 1990 Global Burden of Disease study proposed disability-adjusted life years (DALYs) to measure disease burden. No comprehensive update of disease burden worldwide incorporating a systematic reassessment of disease and injury-specific epidemiology has been done since the 1990 study. We aimed to calculate disease burden worldwide and for 21 regions for 1990, 2005, and 2010 with methods to enable meaningful comparisons over time. METHODS: We calculated DALYs as the sum of years of life lost (YLLs) and years lived with disability (YLDs). DALYs were calculated for 291 causes, 20 age groups, both sexes, and for 187 countries, and aggregated to regional and global estimates of disease burden for three points in time with strictly comparable definitions and methods. YLLs were calculated from age-sex-country-time-specific estimates of mortality by cause, with death by standardised lost life expectancy at each age. YLDs were calculated as prevalence of 1160 disabling sequelae, by age, sex, and cause, and weighted by new disability weights for each health state. Neither YLLs nor YLDs were age-weighted or discounted. Uncertainty around cause-specific DALYs was calculated incorporating uncertainty in levels of all-cause mortality, cause-specific mortality, prevalence, and disability weights. FINDINGS: Global DALYs remained stable from 1990 (2.503 billion) to 2010 (2.490 billion). Crude DALYs per 1000 decreased by 23% (472 per 1000 to 361 per 1000). An important shift has occurred in DALY composition with the contribution of deaths and disability among children (younger than 5 years of age) declining from 41% of global DALYs in 1990 to 25% in 2010. YLLs typically account for about half of disease burden in more developed regions (high-income Asia Pacific, western Europe, high-income North America, and Australasia), rising to over 80% of DALYs in sub-Saharan Africa. In 1990, 47% of DALYs worldwide were from communicable, maternal, neonatal, and nutritional disorders, 43% from non-communicable diseases, and 10% from injuries. By 2010, this had shifted to 35%, 54%, and 11%, respectively. Ischaemic heart disease was the leading cause of DALYs worldwide in 2010 (up from fourth rank in 1990, increasing by 29%), followed by lower respiratory infections (top rank in 1990; 44% decline in DALYs), stroke (fifth in 1990; 19% increase), diarrhoeal diseases (second in 1990; 51% decrease), and HIV/AIDS (33rd in 1990; 351% increase). Major depressive disorder increased from 15th to 11th rank (37% increase) and road injury from 12th to 10th rank (34% increase). Substantial heterogeneity exists in rankings of leading causes of disease burden among regions. INTERPRETATION: Global disease burden has continued to shift away from communicable to non-communicable diseases and from premature death to years lived with disability. In sub-Saharan Africa, however, many communicable, maternal, neonatal, and nutritional disorders remain the dominant causes of disease burden. The rising burden from mental and behavioural disorders, musculoskeletal disorders, and diabetes will impose new challenges on health systems. Regional heterogeneity highlights the importance of understanding local burden of disease and setting goals and targets for the post-2015 agenda taking such patterns into account. Because of improved definitions, methods, and data, these results for 1990 and 2010 supersede all previously published Global Burden of Disease results. FUNDING: Bill & Melinda Gates Foundation.
PMID: 23245608
ISSN: 0140-6736
CID: 209502
Common values in assessing health outcomes from disease and injury: disability weights measurement study for the Global Burden of Disease Study 2010
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.
PMID: 23245605
ISSN: 0140-6736
CID: 209592
The isolation of CHO cells with a site conferring a high and reproducible transgene amplification rate
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.
PMID: 23376841
ISSN: 0168-1656
CID: 524092
B cell TLR7 expression drives anti-RNA autoantibody production and exacerbates disease in systemic lupus erythematosus-prone mice
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.
PMCID:3544945
PMID: 23150717
ISSN: 1550-6606
CID: 2410372
Crystal structure of a voltage-gated K+ channel pore module in a closed state in lipid membranes
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.
PMCID:3522301
PMID: 23095758
ISSN: 0021-9258
CID: 552522
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2012:109(50):20379-84.DOI: 10.1073/pnas.1218052109
In vivo directed differentiation of pluripotent stem cells for skeletal regeneration
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.
PMCID:3528603
PMID: 23169671
ISSN: 0027-8424
CID: 900912
