FRET Biosensors: Engineering Fluorescent Proteins as Biological Tools for Studying Parkinson’s Disease

Parkinson’s Disease (PD) is a common neurodegenerative disease with over 200,000 new cases each year. In general, the cause of the disease is unknown, but oxidative stress inside of neurons has been associated with the disease’s pathology for some time. Currently, techniques to study the onset of PD inside of neurons are limited. This makes treatments and causes difficult to discover. One solution to this has been fluorescent protein biosensors. In short, these proteins can be engineered to glow when a certain state is achieved inside a cell. The present research discusses the engineering of a genetically-encoded fluorescent protein (FP) sensor able to detect reactive oxygen species (peroxide, hydroxyl, superoxide, etc.) inside of neurons, giving one the ability to enhance their understanding of the role these species play in the onset of the disease. This sensor relies on Förster Resonance Energy Transfer (FRET) between a green fluorescent protein and a red fluorescent protein to facilitate red-shifting of the sensor’s emission spectrum. Linked via a short polypeptide chain, the energy transfer efficiency of these combined FPs can vary greatly. Various linker lengths and FP combinations were experimentally tested to draw conclusions about their performance. The current trajectory of the research currently implies that those combinations with the shortest linker lengths will yield the highest-performing sensors. This sensor is another vital piece in the library of tools which can be used to help us begin answering the many questions we have about PD and its pathology

Reduction of Sunburn Damage to Skin by Topical Application of Vitamin E Acetate Following Exposure to Ultraviolet B Radiation: Effect of Delaying Application or of Reducing Concentration of Vitamin E Acetate Applied

The skin of the skh-1 mouse after ultraviolet B (280-320 nm, UVB) irradiation shows the pathological changes typical of sunburn damage: spongiosis (edematous spaces) around some cells, necrosis of keratinocytes, giving rise to sunburn cells, inflammatory infiltration ofpolymorphonuclear leucocytes, etc. In our previous study, these were accompanied by erythema, increased skin sensitivity, and edematous swelling. The topical application of tocopherol acetate (TA) immediately after the UVB exposure decreased these changes. In this paper, multiple measurements of the skin thickness were made at different locations along the magnetic resonance imaging (MRI) cross-sectional image of the skin. This permits effects to be quantified with (if desired) the contralateral half of the back serving as an internal control, either exposed (positive control) or unexposed (negative control). Topical application of TA resulted in an increase in the concentration of free tocopherol in the skin. No qualitative differences in ultrastructural appearance of the DVB-irradiated, TA-treated skin could be discerned by careful examination. In vivo high resolution video microscopy of blood flow in venules of the irradiated mouse ear revealed a large (tenfold) but not statistically significant decrease in stationary lymphocytes adhering to the venule walls. The delaying of the application of TA up to 8 hours after the termination of UVB irradiation still offered statistically significant protection as did immediate application of 5% TA in diluent Myritol 318 (Delios S, Henkel)

Antigenicity and immunogenicity of recombinant envelope glycoproteins of SIVmac32H with different in vivo passage histories.

Shortly after infection of two rhesus monkeys (Macaca mulatta) either with a SIVmac32H challenge stock or with the same virus that had been passaged in another rhesus monkey for 11 months, SIV-envelope genes were cloned from their peripheral blood mononuclear cells and subsequently expressed by recombinant vaccinia viruses. The molecular weights and antigenicities of the thus produced envelope glycoproteins were largely identical to those of the native SIV. The envelope glycoprotein derived from the in vivo passaged virus proved to be poorly recognized by virus neutralizing monoclonal antibodies directed against one of the seven antigenic sites for which monoclonal antibodies were available. Immunization studies in rats showed that this protein was also less efficient in inducing antibodies against this antigenic site, and that it induced significantly lower levels of virus neutralizing antibodies than the other SIV-envelope glycoprotein. The immunogenicity of the SIV-envelope glycoprotein incorporated into immune stimulating complexes (iscoms) was compared to that of the same protein presented with Quil A or MDP-tsl

Next Generation Bipolar Plates for Automotive PEM Fuel Cells

The results of a successful U.S. Department of Energy (DoE) funded two-year $2.9 MM program lead by GrafTech International Inc. (GrafTech) are reported and summarized. The program goal was to develop the next generation of high temperature proton exchange membrane (PEM) fuel cell bipolar plates for use in transportation fuel cell applications operating at temperatures up to 120 °C. The bipolar plate composite developed during the program is based on GrafTech’s GRAFCELL resin impregnated flexible graphite technology and makes use of a high temperature Huntsman Advanced Materials resin system which extends the upper use temperature of the composite to the DoE target. High temperature performance of the new composite is achieved with the added benefit of improvements in strength, modulus, and dimensional stability over the incumbent resin systems. Other physical properties, including thermal and electrical conductivity of the new composite are identical to or not adversely affected by the new resin system. Using the new bipolar plate composite system, machined plates were fabricated and tested in high temperature single-cell fuel cells operating at 120 °C for over 1100 hours by Case Western Reserve University. Final verification of performance was done on embossed full-size plates which were fabricated and glued into bipolar plates by GrafTech. Stack testing was done on a 10-cell full-sized stack under a simulated drive cycle protocol by Ballard Power Systems. Freeze-thaw performance was conducted by Ballard on a separate 5-cell stack and shown to be within specification. A third stack was assembled and shipped to Argonne National Laboratory for independent performance verification. Manufacturing cost estimate for the production of the new bipolar plate composite at current and high volume production scenarios was performed by Directed Technologies Inc. (DTI). The production cost estimates were consistent with previous DoE cost estimates performed by DTI for the DoE on metal plates. The final result of DTI’s analysis for the high volume manufacturing scenario ($6.85 /kW) came in slightly above the DoE target of $3 to$5/kW. This estimate was derived using a “Best Case Scenario” for many of the production process steps and raw material costs with projections to high volumes. Some of the process improvements assumed in this “Best Case Scenario” including high speed high impact forming and solvent-less resins, have not yet been implemented, but have a high probability of potential success

The extrusion of Ti-6Al-4V

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Differential white cell counts by frequency distribution analysis of cell volumes

Absolute neutrophil and lymphocyte counts on peripheral blood can be made by analysis of the output from a Coulter particle counter, utilizing the difference in the relative cell volume between these two types of cell. A comparison has been made between the results obtained by volume analysis and those obtained by standard microscopical techniques in 10 normal people and 45 patients. The absolute neutrophil count obtained by volume analysis agreed well with values obtained by microscopy; the lymphocyte count did not give such good agreement, since the smaller number of cells counted gave rise to larger sampling errors. The method of volume analysis is suitable for the assessment of absolute neutrophil counts for clinical use