Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics.

Developing robust methodology for the sustainable production of red blood cells in vitro is essential for providing an alternative source of clinical-quality blood, particularly for individuals with rare blood group phenotypes. Immortalized erythroid progenitor cell lines are the most promising emergent technology for achieving this goal. We previously created the erythroid cell line BEL-A from bone marrow CD34+ cells that had improved differentiation and enucleation potential compared to other lines reported. In this study we show that our immortalization approach is reproducible for erythroid cells differentiated from bone marrow and also from far more accessible peripheral and cord blood CD34+ cells, consistently generating lines with similar improved erythroid performance. Extensive characterization of the lines shows them to accurately recapitulate their primary cell equivalents and provides a molecular signature for immortalization. In addition, we show that only cells at a specific stage of erythropoiesis, predominantly proerythroblasts, are amenable to immortalization. Our methodology provides a step forward in the drive for a sustainable supply of red cells for clinical use and for the generation of model cellular systems for the study of erythropoiesis in health and disease, with the added benefit of an indefinite expansion window for manipulation of molecular targets.

Lethal trap trees: a potential option for emerald ash borer (Agrilus planipennis Fairmaire) management.

BACKGROUND: Economic and ecological impacts of ash (Fraxinus spp.) mortality resulting from emerald ash borer (EAB) (Agrilus planipennis Fairmaire) invasion are severe in forested, residential and urban areas. Management options include girdling ash trees to attract ovipositing adult beetles and then destroying infested trees before larvae develop or protecting ash with a highly effective, systemic emamectin benzoate insecticide. Injecting this insecticide and then girdling injected trees a few weeks later could effectively create lethal trap trees, similar to a bait-and-kill tactic, if girdling does not interfere with insecticide translocation. We compared EAB larval densities on girdled trees, trees injected with the emamectin benzoate insecticide, trees injected with the insecticide and then girdled 18-21 days later and untreated controls at multiple sites. RESULTS: Pretreatment larval densities did not differ among treatments. Current-year larval densities were higher on girdled and control trees than on any trees treated with insecticide at all sites. Foliar residue analysis and adult EAB bioassays showed that girdling trees after insecticide injections did not reduce insecticide translocation. CONCLUSIONS: Girdling ash trees to attract adult EAB did not reduce efficacy of emamectin benzoate trunk injections applied ≥ 18 days earlier and could potentially be used in integrated management programs to slow EAB population growth.

Efficacy of imidacloprid, trunk-injected into Acer platanoides, for control of adult Asian longhorned beetles (Coleoptera: Cerambycidae).

Feeding experiments with Asian longhorned beetles (Anoplophora glabripennis (Motschulsky)) in a quarantine laboratory were used to assess the effectiveness of imidacloprid in reducing adult fecundity and survival. The beetles were fed twigs and leaves cut between June-September 2010 from Norway maples (Acer platanoides L.) in the beetle-infested area of Worcester, MA. Treated trees had been trunk-injected once with imidacloprid in spring 2010 under the U.S. Department of Agriculture-Animal and Plant Health Inspection Service operational eradication program. The 21 d LC50 value for adult beetles feeding on twig bark from imidacloprid-injected trees was 1.3 ppm. Adult reproductive output and survival were significantly reduced when beetles fed on twig bark or leaves from treated trees. However, results varied widely, with many twig samples having no detectable imidacloprid and little effect on the beetles. When twigs with > 1 ppm imidacloprid in the bark were fed to mated beetles, the number of larvae produced was reduced by 94% and median adult survival was reduced to 14 d. For twigs with < 1 ppm imidacloprid, 68% of reproductively mature mated beetles survived 21 d and 56% of unmated recently eclosed beetles survived 42 d. For twigs with < 1 ppm, beetles ingested an average of 30 nanograms of imidacloprid per day. Bark consumption was reduced at higher imidacloprid levels (> 1 ppm). When given a choice of control twigs and twigs from injected trees, beetles did not show a strong preference.

Health insurance sources for nonelderly patient visits to physician offices, hospital outpatient departments, and emergency departments in the United States.

The authors used data from nationally representative surveys to estimate health insurance sources for non-elderly patient visits to U.S. physicians. Results show that hospital emergency departments attract a greater share of ambulatory care visits by uninsured patients than by patients with either Medicaid or private insurance. Results also show that hospital outpatient departments attract a greater share of visits by uninsured patients or patients with Medicaid than by patients with private insurance. The annual visit rate of uninsured individuals for nonemergency care is less than half of that for individuals with either private insurance or Medicaid. The proportion of uninsured emergency department visits by individuals between the ages of 0 and 64 years was significantly greater than the proportion of uninsured individuals between the ages of 0 and 64 years. In contrast, the proportion of uninsured physician office visits by individuals between the ages of 0 and 64 years was significantly less than the proportion of uninsured individuals between the ages of 0 and 64 years.