Genetic engineering of megakaryocytes from blood progenitor cells using mRNA lipid nanoparticles.

BACKGROUND: Platelets are an essential component of hemorrhage control and management, and engineering platelets to express therapeutic proteins could expand their use as a cell therapy. Genetically engineered platelets can be achieved by modifying the platelet precursor cells, megakaryocytes (MKs). Current strategies include transfecting MK progenitors ex vivo with viral vectors harbouring lineage-driven transgenes and inducing the production of "in vitro" modified platelets. The use of viruses, however, poses challenges in clinical implementation, and no methods currently exist to genetically modify MKs with non-viral techniques. Lipid nanoparticles (LNP) are a non-viral delivery system that could enable a facile strategy to modify MKs with a variety of nucleic acid payloads. OBJECTIVE: To investigate whether LNP can transfect cultured hematopoietic stem/progenitor cell (HSPC)-derived MKs to express exogenous proteins and induce functional changes. METHOD: MK and MK progenitors differentiated from cord-blood derived HSPCs were treated with lipid nanoparticle formulations containing mRNA and resembling the clinically approved LNP formulations. Transfection efficiency was assessed through flow cytometry by expression of enhanced green fluorescent protein. Functional changes to the MKs were assessed through rotational thromboelastometry by expression of exogenous coagulation factor VII (FVII), a representative physiologically relevant protein. RESULT: LNP enabled transfection efficiencies of 99% in MKs, and did not impair MK maturation, viability, and morphology. MKs engineered to express exogenous FVII decreased clotting time in FVII-deficient plasma following clot initiation. CONCLUSION: This approach provides an easy-to-use modular platform to genetically modify MK and MK progenitors, which can be potentially extended to producing genetically modified cultured platelets.

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells.

T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains an important challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergized to activate notch signaling in nascent hematopoietic stem and progenitor cells, and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimized medium formulations that enabled efficient and chemically defined maturation of functional CD8αß+, CD4-, CD3+, TCRαß+ T cells with a diverse TCR repertoire.

Additions to the knowledge of Nevada carabid beetles (Coleoptera: Carabidae) and a preliminary list of carabids from the Great Basin National Park.

BACKGROUND: Additions to the list of Carabidae known for Nevada, USA and carabid beetles found in the Great Basin National Park, NV are reported with notes on ecology and identification resources. NEW INFORMATION: For 79 species of carabids, we present 57 new state records, two state records previously reported in online resources, one confirmation of a previous questionable record for the state, and report 22 records for the Great Basin National Park that includes three new state records.

Characterization of gamma-alumina-supported manganese oxide as an incineration catalyst for trichloroethylene.

Trichloroethylene (TCE) decomposition over a MnOx/ gamma-Al2O3 catalyst in a fixed-bed reactor was conducted in this study. The MnOx/gamma-Al2O3 powders were prepared by the incipient wetness impregnation method with aqueous solution of manganese nitrate. The catalysts were characterized by DTA-TGA, XRD, porosity analysis, SEM, EDX, and XPS. The results show that the main distinct weight loss is found at the temperature around 373 and 873 K,the MnO peaks (2theta = 34.9 degrees and 40.5 degrees) are only observed crystal phase on the fresh catalyst, the SEM image of the MnOx-impregnated gamma-Al2O3 support is much different from the calcined catalyst, and the Mn element quantity on the catalyst surface is higher than that of the impregnated support. The products and reactants distributions from the oxidation of TCE over MnOx/gamma-Al2O3 were analyzed by GC. The results show that the TCE conversion starts from 5% at 443 K and rises to very high values in the 673-873 K ranges and that the CO2 yield also pushes to 99% at the same temperature ranges. HCl and Cl2 are the other main products with little halogenated VOC intermediates.

The catalytic incineration of trichloroethylene over a gamma-alumina supported manganese oxide catalyst.

TCE decomposition over a Mn2O3/gamma-Al2O3 catalyst in a fixed bed reactor was conducted in this study. Preliminarily, three catalysts including Mn2O3/gamma-Al2O3, NiO/gamma-Al2O3, and Pt/gamma-Al2O3 were used to incinerate TCE and the results show that the Mn2O3/gamma-Al2O3 catalyst has the best performance. The effects of operating parameters, such as inlet temperature, space velocity, TCE inlet concentration, and oxygen concentration on the catalytic incineration of TCE over the Mn2O3/gamma-Al2O3 catalyst were then performed. The results show that conversion of TCE increases as inlet temperature and oxygen concentration increase, and decreases with the increases of TCE concentration and space velocity. The activity of the catalyst decreases significantly while TCE incineration is operated under a low temperature, 365 degrees C. However, the activity of the catalyst does not change much while the operating temperature is as high as 500 degrees C. The catalysts were characterized by the surface and pore size analysis, XRD, XPS, EDS, and SEM before and after the tests. The results show that the catalytic crystal is Mn2O3, the catalytic deactivation is not due to carbonaceous material, and the chlorine element is adsorbed on the surface of catalysts.