Crude subcellular fractionation of cultured mammalian cell lines

<p>Abstract</p> <p>Background</p> <p>The expression and study of recombinant proteins in mammalian culture systems can be complicated during the cell lysis procedure by contaminating proteins from cellular compartments distinct from those within which the protein of interest resides and also by solubility issues that may arise from the use of a single lysis buffer. Partial subcellular fractionation using buffers of increasing stringency, rather than whole cell lysis is one way in which to avoid or reduce this contamination and ensure complete recovery of the target protein. Currently published protocols involve time consuming centrifugation steps which may require expensive equipment and commercially available kits can be prohibitively expensive when handling large or multiple samples.</p> <p>Findings</p> <p>We have established a protocol to sequentially extract proteins from cultured mammalian cells in fractions enriched for cytosolic, membrane bound organellar, nuclear and insoluble proteins. All of the buffers used can be made inexpensively and easily and the protocol requires no costly equipment. While the method was optimized for a specific cell type, we demonstrate that the protocol can be applied to a variety of commonly used cell lines and anticipate that it can be applied to any cell line via simple optimization of the primary extraction step.</p> <p>Conclusion</p> <p>We describe a protocol for the crude subcellular fractionation of cultured mammalian cells that is both straightforward and cost effective and may facilitate the more accurate study of recombinant proteins and the generation of purer preparations of said proteins from cell extracts.</p

Ouachita Baptist University Founder\u27s Day

This is the program for the faculty recital celebrating the Ouachita Baptist University\u27s Founder\u27s Day and for the unveiling of the bust of Dr. J. W. Conger. In addition to performances by Dr. William Horton and Amelia Carter, Dr. Vester Wolber provided an invocation, OBU President Ralph Phelps, Jr. gave a welcome address and recognized the trustees and other distinguished guests, and Dr. Marvin Green gave a benediction. This ceremony took place on February 21, 1967

Book Reviews

Reviews of the following books: Acadia, Maine, and New Scotland: Marginal Colonies in the Seventeenth Century by John G. Reid; Faces of Maine by Bob Niss; Morning Was Starlight: My Maine Boyhood by Ernest Dodge

Participatory Climate Research in a Dynamic Urban Context: Activities of the Consortium for Climate Risk in the Urban Northeast (CCRUN)

The Consortium for Climate Risk in the Urban Northeast (CCRUN), one of ten NOAA-RISAs, supports resilience efforts in the urban corridor stretching from Philadelphia to Boston. Challenges and opportunities include the diverse set of needs in broad urban contexts, as well as the integration of interdisciplinary perspectives. CCRUN is addressing these challenges through strategies including: 1) the development of an integrated project framework, 2) stakeholder surveys, 3) leveraging extreme weather events as focusing opportunities, and 4) a seminar series that enables scientists and stakeholders to partner. While recognizing that the most extreme weather events will always lead to surprises (even with sound planning), CCRUN endeavors to remain flexible by facilitating place-based research in an interdisciplinary context

Low temperature reduction of hexavalent chromium by a microbial enrichment consortium and a novel strain of Arthrobacter aurescens

BACKGROUND: Chromium is a transition metal most commonly found in the environment in its trivalent [Cr(III)] and hexavalent [Cr(VI)] forms. The EPA maximum total chromium contaminant level for drinking water is 0.1 mg/l (0.1 ppm). Many water sources, especially underground sources, are at low temperatures (less than or equal to 15 Centigrade) year round. It is important to evaluate the possibility of microbial remediation of Cr(VI) contamination using microorganisms adapted to these low temperatures (psychrophiles). RESULTS: Core samples obtained from a Cr(VI) contaminated aquifer at the Hanford facility in Washington were enriched in Vogel Bonner medium at 10 Centigrade with 0, 25, 50, 100, 200, 400 and 1000 mg/l Cr(VI). The extent of Cr(VI) reduction was evaluated using the diphenyl carbazide assay. Resistance to Cr(VI) up to and including 1000 mg/l Cr(VI) was observed in the consortium experiments. Reduction was slow or not observed at and above 100 mg/l Cr(VI) using the enrichment consortium. Average time to complete reduction of Cr(VI) in the 30 and 60 mg/l Cr(VI) cultures of the consortium was 8 and 17 days, respectively at 10 Centigrade. Lyophilized consortium cells did not demonstrate adsorption of Cr(VI) over a 24 hour period. Successful isolation of a Cr(VI) reducing organism (designated P4) from the consortium was confirmed by 16S rDNA amplification and sequencing. Average time to complete reduction of Cr(VI) at 10 Centigrade in the 25 and 50 mg/l Cr(VI) cultures of the isolate P4 was 3 and 5 days, respectively. The 16S rDNA sequence from isolate P4 identified this organism as a strain of Arthrobacter aurescens, a species that has not previously been shown to be capable of low temperature Cr(VI) reduction. CONCLUSION: A. aurescens, indigenous to the subsurface, has the potential to be a predominant metal reducer in enhanced, in situ subsurface bioremediation efforts involving Cr(VI) and possibly other heavy metals and radionuclides

Experimental Testing and Modeling of 5 kW Oil-Free Open Drive Scroll Expander Using R245fa

Organic Rankine Cycles (ORC) are thermodynamic power cycles designed to generate work from low temperature sources, typically between 80 °C to 270 °C.  The low temperature heat input makes this technology attractive for applications in waste heat recovery from industrial processes, exhaust gas from diesel engines, solar systems, geothermal systems, and others.  The expander has the greatest effect on increasing the efficiency of an ORC. The operating  conditions that the expander is subjected to are directed related to its efficiency.  The performance of a 5 kW scroll expander with a displacement of 73.6 cm3 per revolution, operating at speeds from 500 to 3600 rpm, and using R245fa as the working fluid in a traditional Organic Ranking cycle is experimentally investigated In this paper. Tests were conducted varying the mass flow rate of the working fluid and varying source temperatures, while measuring the effective expander power production and the isentropic efficiency.  The experimental data was then used to develop a model of the scroll expander. Using this model, thermodynamic simulations were carried out for applications similar to exhaust gas waste heat recovery from internal combustion engines

Primary Arthrodesis for Diabetic Ankle Fractures

A grant from the One-University Open Access Fund at the University of Kansas was used to defray the author's publication fees in this Open Access journal. The Open Access Fund, administered by librarians from the KU, KU Law, and KUMC libraries, is made possible by contributions from the offices of KU Provost, KU Vice Chancellor for Research & Graduate Studies, and KUMC Vice Chancellor for Research. For more information about the Open Access Fund, please see http://library.kumc.edu/authors-fund.xml.Background: Treatment of ankle fractures in patients with diabetes is associated with increased complication rates. Ankle arthrodesis is considered a salvage procedure after failed ankle fracture fixation, yet primary ankle arthrodesis has been proposed as a treatment option for patients with significant diabetes-related complications. To date, the characteristics of patients who undergo primary ankle arthrodesis and the associated outcomes have not been described. Methods: A retrospective review was performed of 13 patients with diabetes who underwent primary arthrodesis for traumatic ankle fracture. Patient demographics were characterized in addition to their diabetes complications, Adelaide Fracture in the Diabetic Ankle (AFDA) score, and fracture type. Outcomes assessed included reoperation rates, infection rates, wound complications, nonunion/malunion, amputation, and development of Charcot arthropathy postoperatively. Results: Patients who underwent primary arthrodesis had high rates of diabetes complications, average AFDA scores of 6.4, and high rates of severe injuries, including 38.5% open fractures and 69.2% fracture dislocations. The overall complication rate for primary arthrodesis of ankle fractures in diabetes patients was more than 75% in this cohort. Complications included a 38.5% reoperation rate, 38.5% infection rate, 53.8% wound complication rate, and 23.1% amputation rate. Despite a high nonunion rate at the attempted fusion sites, 89.9% of fractures healed and patients had a stable extremity. Conclusion: This review is the first to characterize the epidemiology and complications of diabetes patients undergoing primary ankle arthrodesis for ankle fractures. In this cohort, patients with multiple diabetic complications and severe injuries underwent primary arthrodesis, which led to an overall high complication rate. Further research is needed to determine the appropriate treatment option for these high-risk patients, and tibiotalocalcaneal stabilization without arthrodesis may be beneficial

Climate Change in New York State Updating the 2011 ClimAID Climate Risk Information Supplement to NYSERDA Report 11-18 (Responding to Climate Change in New York State)

In its 2013-2014 Fifth Assessment Report (AR5), the Intergovernmental Panel on Climate Change (IPCC) states that there is a greater than 95 percent chance that rising global average temperatures, observed since the mid-20th century, are primarily due to human activities. As had been predicted in the 1800s, the principal driver of climate change over the past century has been increasing levels of atmospheric greenhouse gases associated with fossil-fuel combustion, changing land-use practices, and other human activities. Atmospheric concentrations of the greenhouse gas carbon dioxide are now approximately 40 percent higher than in preindustrial times. Concentrations of other important greenhouse gases, including methane and nitrous oxide, have increased rapidly as well

Additively Manufactured RCS for Small Satellites and Landers

After a fifty year absence, NASA’s return to the lunar surface under the Artemis Program – for long term human exploration and utilization – is driving commercial and academic opportunities for small satellite and small lander platforms (e.g., Commercial Lunar Payload Services program – CLPS). Bipropellant thrusters are a reliable, low risk, and flight proven method for the propulsion and attitude control that is required for complex maneuvers such entry, descent, and landing (EDL) or in-space proximity operations. However, due to the increasingly competitive commercial spaceflight market in the last decade, satellite subsystems must also be affordable to buy their way into the final mission design and engineering solution. Therefore starting in 2019, and based off prior satellite integration work, Aerojet Rocketdyne (AR) undertook an advanced propulsion development effort to combine modern metal additive manufacturing (AM) techniques with thrust scalable hypergolic MON-25 propulsion technology to create a high performance and fully integrated (i.e., multiple thrusters integrated into a single package) reaction control system (RCS) at a fraction of the production cost when compared to the heritage designs that are assembled from individual thrusters. The point-of-departure for the RCS design comes from a new line of additively manufactured thrusters that stably burn volatile MON-25 oxidizer with monomethylhydrazine (MMH) fuel at thrust levels of 5 lbf and 100 lbf. Cost at the subsystem level is lowered by the AM integration of parts and functions which reduces the build of materials, touch labor, and assembly time. In addition, AM allows the design to be adaptable to changing requirements such as the number of thrusters, orientation, and thrust level. Cost at the satellite level is reduced by leveraging MON-25’s lower freezing point of -55 °C (compared to traditional dinitrogen tetroxide oxidizer) to minimize mass, thermal, and power requirements while operating in deep-space environments. In addition, thruster operation at the equal volume mixture ratio for MMH/MON-25 allows for a modular approach to tank design and a predictable center of gravity during maneuvering. This paper provides an overview of the ISE-5 and the ISE-100 MON-25 thruster technology that powers the integrated designs as well as the development progress of the AM RCS concept itself. This includes reduction to practice activities such as proof-of-concept AM material test demonstrators and water flow test units

Probability of detection of defects in coatings with electronic shearography

The goal of this research was to utilize statistical methods to evaluate the probability of detection (POD) of defects in coatings using electronic shearography. The coating system utilized in the POD studies was to be the paint system currently utilized on the external casings of the NASA Space Transportation System (STS) Revised Solid Rocket Motor (RSRM) boosters. The population of samples was to be large enough to determine the minimum defect size for 90 percent probability of detection of 95 percent confidence POD on these coatings. Also, the best methods to excite coatings on aerospace components to induce deformations for measurement by electronic shearography were to be determined