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Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.

Extracellular vesicles are nanoparticles produced by cells. They are composed of cellular membrane with associated membrane proteins that surrounds an aqueous core containing soluble molecules such as proteins and nucleic acids, like miRNA and mRNA. They are important in many physiological and pathological processes as they can transfer biological molecules from producer cells to acceptor cells. Preparation of the niche for cancer metastasis, stimulation of tissue regeneration and orchestration of the immune response are examples of the diverse processes in which extracellular vesicles have been implicated. As a result, these vesicles have formed a source of inspiration for many scientific fields. They could be used, for example, as liquid biopsies in diagnostics, as therapeutics in regenerative medicine, or as drug delivery vehicles for transport of medicines. In this Account, we focus on drug delivery applications. As we learn more and more about these vesicles, the complexity increases. What originally appeared to be a relatively uniform population of cellular vesicles is increasingly subdivided into different subsets. Cells make various distinct vesicle types whose physicochemical aspects and composition is influenced by parental cell type, cellular activation state, local microenvironment, biogenesis pathway, and intracellular cargo sorting routes. It has proven difficult to assess the effects of changes in production protocol on the characteristics of the cell-derived vesicle population. On top of that, each isolation method for vesicles necessarily enriches certain vesicle classes and subpopulations while depleting others. Also, each method is associated with a varying degree of vesicle purity and concomitant coisolation of nonvesicular material. What emerges is a staggering heterogeneity. This constitutes one of the main challenges of the field as small changes in production and isolation protocols may have large impact on the vesicle characteristics and

Daniel E Murphy,1,2 Ralph J Panos1,2 1Pulmonary, Critical Care and Sleep Medicine Division, Cincinnati Veterans Affairs Medical Center, 2Pulmonary, Critical Care and Sleep Medicine Division, University of Cincinnati College of Medicine, Cincinnati, OH, USA Abstract: Chronic obstructive pulmonary disease (COPD) is frequently under-recognized and underdiagnosed. To determine the natural history of recognized and unrecognized COPD, we studied the rate of diagnosis, health care utilization, and mortality in patients with airflow limitation (AFL). Three hundred forty-seven outpatients at the Cincinnati Veterans Administration Medical Center performed spirometry and completed a respiratory questionnaire. Patients were followed for a minimum of 30 months and medical records were reviewed for COPD diagnosis, mortality, respiratory-related health care utilization, comorbidities, and respiratory medications. Three hundred twenty-five of 347 (94%) patients performed technically adequate spirometry and completed questionnaires. When AFL was defined by fixed ratio (FR, forced expiratory volume in 1 second [FEV1]/forced vital capacity [FVC] < 0.7), patients with AFL and a diagnosis of COPD had a higher annual mortality rate (7.1% ± 2% versus 2.4% ± 0.8%, P = 0.01), more hospitalizations per year (0.2 ± 0.06 versus 0.04 ± 0.01, P < 0.001 mean ± standard error of the mean), increased respiratory symptoms (12.0 ± 0.9 versus 7.2 ± 0.6, P < 0.0001), and higher Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage compared with undiagnosed patients. Ninety-two of 137 patients with AFL (67%) had unrecognized AFL; 16 (17%) of the 92 were subsequently diagnosed. When AFL was defined by the lower limit of normal (LLN, FEV1/FVC < LLN), 67 of 103 patients (65%) had unrecognized AFL; 12 (18%) of the 67 were subsequently diagnosed. Patients with AFL defined by FR who were subs

This thesis is an analysis of U.S. policy toward Iraq since the Gulf War. UN Security Council Resolution 687 was the formal cease-fire agreement ending the Gulf War, the resolution requiring the elimination of Iraqi weapons of mass destruction and created the UN Special Commission (UNSCOM). Attempts to gain Iraqi compliance with Resolution 687 consumed U.S. Iraq policy for nine years. In 1999, UNSCOM was disestablished without fulfilling its mandate. The Security Council then adopted Resolution 1284 in attempt to introduce a new inspection regime into Iraq. This thesis examines the factors required to successfully compel Iraqi compliance with Resolution 687 and now Resolution 1284. The findings of this research conclude that current U.S. policy toward Iraq does not contain elements needed to successfully compel Iraqi compliance. The thesis then offers three policy options to deal with Iraq. The advantages and disadvantages of each are discussed. The thesis concludes that although current policy does not support the re-entry of a viable inspection regime, current policy does support the overall U.S. objective of containing Iraq. It is argued that this policy should be maintained in the near-term while the United States fully develops regime change as a long-term strategy.

This thesis is an analysis of U.S. policy toward Iraq since the Gulf War. UN Security Council Resolution 687 was the formal cease-fire agreement ending the Gulf War, the resolution requiring the elimination of Iraqi weapons of mass destruction and created the UN Special Commission (UNSCOM). Attempts to gain Iraqi compliance with Resolution 687 consumed U.S. Iraq policy for nine years. In 1999, UNSCOM was disestablished without fulfilling its mandate. The Security Council then adopted Resolution 1284 in attempt to introduce a new inspection regime into Iraq. This thesis examines the factors required to successfully compel Iraqi compliance with Resolution 687 and now Resolution 1284. The findings of this research conclude that current U.S. policy toward Iraq does not contain elements needed to successfully compel Iraqi compliance. The thesis then offers three policy options to deal with Iraq. The advantages and disadvantages of each are discussed. The thesis concludes that although current policy does not support the re-entry of a viable inspection regime, current policy does support the overall U.S. objective of containing Iraq. It is argued that this policy should be maintained in the near-term while the United States fully develops regime change as a long-term strategy.

This thesis is an analysis of U.S. policy toward Iraq since the Gulf War. UN Security Council Resolution 687 was the formal cease-fire agreement ending the Gulf War, the resolution requiring the elimination of Iraqi weapons of mass destruction and created the UN Special Commission (UNSCOM). Attempts to gain Iraqi compliance with Resolution 687 consumed U.S. Iraq policy for nine years. In 1999, UNSCOM was disestablished without fulfilling its mandate. The Security Council then adopted Resolution 1284 in attempt to introduce a new inspection regime into Iraq. This thesis examines the factors required to successfully compel Iraqi compliance with Resolution 687 and now Resolution 1284. The findings of this research conclude that current U.S. policy toward Iraq does not contain elements needed to successfully compel Iraqi compliance. The thesis then offers three policy options to deal with Iraq. The advantages and disadvantages of each are discussed. The thesis concludes that although current policy does not support the re-entry of a viable inspection regime, current policy does support the overall U.S. objective of containing Iraq. It is argued that this policy should be maintained in the near-term while the United States fully develops regime change as a long-term strategy., http://archive.org/details/respondingtosadd109459302, U.S. Navy (U.S.N.) author., Approved for public release; distribution is unlimited.