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Effects of amino acid substitutions on the biological activity of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori)
Highlights: • Amino acid substitutions modulate Fc-mediated functions of glyco-engineered mAbs. • Fc-engineered mAbs produced by silkworms show unique Fc-mediated functions. • Transgenic silkworm is a promising system for the production of Fc-engineered mAbs. Recombinant monoclonal antibodies (mAbs) have been used in various therapeutic applications including cancer therapy. Fc-mediated effector functions play a pivotal role in the tumor-killing activities of some tumor-targeting mAbs, and Fc-engineering technologies with glyco-engineering or amino acid substitutions at the antibody Fc region have been used to enhance cytotoxic activities including antibody-dependent cellular cytotoxicity (ADCC). We previously reported that the mAbs produced using transgenic silkworms showed stronger ADCC activity and lower complement-dependent cytotoxicity (CDC) activity than mAbs derived from Chinese hamster ovary (CHO) cells due to their unique N-glycan structure (lack of core-fucose and non-reducing terminal galactose). In this study, we generated anti-CD20 mAbs with amino acid substitutions using transgenic silkworms and analyzed their biological activities to assess the effect of the combination of glyco-engineering and amino acid substitutions on the Fc-mediated function of mAbs. Three types of amino acid substitutions at the Fc region (G236A/S239D/I332E, L234A/L235A, and K326W/E333S) modified the Fc-mediated biological activities of silkworm-derived mAbs as in the case of CHO-derived mAbs, resulting in the generation of Fc-engineered mAbs with characteristic Fc-mediated functions. The combination of amino acid substitutions at the Fc region and glyco-engineering using transgenic silkworm made it possible to generate Fc-engineered mAbs with suitable Fc-mediated biological functions depending on the pharmacological mechanism of their actions. Transgenic silkworms were shown to be a promising system for the production of Fc-engineered mAbs.
Activation of human natural killer cells by the soluble form of cellular prion protein
Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.
Fluorocarbon based atomic layer etching of Si{sub 3}N{sub 4} and etching selectivity of SiO{sub 2} over Si{sub 3}N{sub 4}
Angstrom-level plasma etching precision is required for semiconductor manufacturing of sub-10 nm critical dimension features. Atomic layer etching (ALE), achieved by a series of self-limited cycles, can precisely control etching depths by limiting the amount of chemical reactant available at the surface. Recently, SiO{sub 2} ALE has been achieved by deposition of a thin (several Angstroms) reactive fluorocarbon (FC) layer on the material surface using controlled FC precursor flow and subsequent low energy Ar{sup +} ion bombardment in a cyclic fashion. Low energy ion bombardment is used to remove the FC layer along with a limited amount of SiO{sub 2} from the surface. In the present article, the authors describe controlled etching of Si{sub 3}N{sub 4} and SiO{sub 2} layers of one to several Angstroms using this cyclic ALE approach. Si{sub 3}N{sub 4} etching and etching selectivity of SiO{sub 2} over Si{sub 3}N{sub 4} were studied and evaluated with regard to the dependence on maximum ion energy, etching step length (ESL), FC surface coverage, and precursor selection. Surface chemistries of Si{sub 3}N{sub 4} were investigated by x-ray photoelectron spectroscopy (XPS) after vacuum transfer at each stage of the ALE process. Since Si{sub 3}N{sub 4} has a lower physical sputtering energy threshold than SiO{sub 2}, Si{sub 3}N{sub 4} physical sputtering can take place after removal of chemical etchant at the end of each cycle for relatively high ion energies. Si{sub 3}N{sub 4} to SiO{sub 2} ALE etching selectivity was observed for these FC depleted conditions. By optimization of the ALE process parameters, e.g., low ion energies, short ESLs, and/or high FC film deposition per cycle, highly selective SiO{sub 2} to Si{sub 3}N{sub 4} etching can be achieved for FC accumulation conditions, where FC can be selectively accumulated on Si{sub 3}N{sub 4} surfaces. This highly selective etching is explained by a lower carbon consumption of Si{sub 3}N{sub 4} as compared to SiO{sub 2}. The comparison of C{sub 4}F{sub 8} and CHF{sub 3} only showed a difference in etching selectivity for FC depleted conditions. For FC accumulation conditions, precursor chemistry has a weak impact on etching selectivity. Surface chemistry analysis shows that surface fluorination and FC reduction take place during a single ALE cycle for FC depleted conditions. A fluorine rich carbon layer was observed on the Si{sub 3}N{sub 4} surface after ALE processes for which FC accumulation takes place. The angle resolved-XPS thickness calculations confirmed the results of the ellipsometry measurements in all cases.
SU-F-T-430: Validation of IBEAM Evo Couch Top for Different Relative Electron Density (RED) Combination During Photon Beam Dose Calculation in Monaco− Treatment Planning System
Purpose: Validation of iBEAM™ evo couch-top for different relative electron density (RED) combination during photon beam dose calculation in Monaco− TPS. Methods: The iBEAM™ evo couch-top has two layers:outer carbon fiber (CF) and inner foam core (FC). To study the beam intensity attenuation of couch-top, measured doses were compared with doses calculated for different REDs. Measurements were performed in solid water phantom with PTW-0.125cc ion-chamber positioned at center of the phantom with 5.3cm thickness slabs placed above and below the chamber. Similarly, in TPS, iBEAM™ evo couch-top was simulated and doses were calculated for different RED combinations (0.2CF-0.2FC, 0.4CF-0.2FC, 0.6CF-0.2FC, 0.8CF-0.2FC, and 1.0CF-0.2FC) by using Monte Carlo dose calculation algorithm in Monaco TPS (V5.1). Doses were measured for every 10 degree gantry angle separation, 10×10cm{sup 2} field size and 6MV photons. Then, attenuation is defined as the ratio of output at posterior gantry angle to output of its opposed anterior gantry angle (e.g.225°/45°). output fluctuation with different gantry angle was within ±0.21%. To confirm above results, dose-planes were measured for five pelvic VMAT plans (360°arc) in PTW two-dimensional array and compared with different calculated dose-planes of above-mentioned couch REDs. Gamma pass rates<1.00) were analyzed for 3%/2mm criteria. Results: Measured and calculated attenuation was in good agreement for the RED combination of 0.2CF-0.2FC and difference was within ±0.515%. However, other density combination showed difference of ±0.9841%, ±1.667%, ±2.9241% and ±2.8832% for 0.4CF-0.2FC, 0.6CF-0.2FC, 0.8CF-0.2FC, and 1.0CF-0.2FC, respectively. Maximum couch-top attenuation was observed at 110°–120° and 240°–250° and decreases linearly as the gantry angle approaches 180°. Moreover, gamma pass rate confirmed the above results and showed maximum pass rate of 96.23% for 0.2CF-0.2FC, whereas others were 95.72%, 95.12%, 94.31% and 93.24%. Conclusion: RED value of 0.2CF-0.2FC was found to be suitable for accurate couch-top modeling for 6MV photon beam Monte Carlo calculations in Monaco TPS.
Characterizing Fluorocarbon Assisted Atomic Layer Etching of Si Using Cyclic Ar/C 4 F 8 and Ar/CHF 3 Plasma
With the increasing interest in establishing directional etching methods capable of atomic scale resolution for fabricating highly scaled electronic devices, the need for development and characterization of atomic layer etching (ALE) processes, or generally etch processes with atomic layer precision, is growing. In this work, a flux-controlled cyclic plasma process is used for etching of SiO 2 and Si at the Angstrom-level. This is based on steady-state Ar plasma, with periodic, precise injection of a fluorocarbon (FC) precursor (C 4 F 8 and CHF 3 ), and synchronized, plasma-based Ar+ ion bombardment [D. Metzler et al., J Vac Sci Technol A 32, 020603 (2014), and D. Metzler et al., J Vac Sci Technol A 34, 01B101 (2016)]. For low energy Ar+ ion bombardment conditions, physical sputter rates are minimized, whereas material can be etched when FC reactants are present at the surface. This cyclic approach offers a large parameter space for process optimization. Etch depth per cycle, removal rates, and self-limitation of removal, along with material dependence of these aspects, were examined as a function of FC surface coverage, ion energy, and etch step length using in situ real time ellipsometry. The deposited FC thickness per cycle is found to have a strong impact on etch depth per cycle of SiO 2 and Si, but is limited with regard to control over material etching selectivity. Ion energy over the 20 to 30 eV range strongly impacts material selectivity. The choice of precursor can have a significant impact on the surface chemistry and chemically enhanced etching. CHF 3 has a lower FC deposition yield for both SiO 2 and Si, and also exhibits a strong substrate dependence of FC deposition yield, in contrast to C4F 8 . The thickness of deposited FC layers using CHF 3 is found to be greater for Si than for SiO 2 . X-ray photoelectron spectroscopy was used to study surface chemistry. When thicker FC films of 11 Å are employed, strong changes of FC film chemistry during a cycle are seen whereas the chemical state of the substrate varies much less. On the other hand, for FC film deposition of 5 Å for each cycle, strong substrate surface chemical changes are seen during an etching cycle. The nature of this cyclic etching with periodic deposition of thin FC films differs significantly from conventional etching with steady-state FC layers since surface conditions change strongly throughout each cycle.
Automotive fuel cell stack and system efficiency and fuel consumption based on vehicle testing on a chassis dynamometer at minus 18 °C to positive 35 °C temperatures
In this work we present an in-depth laboratory technology assessment of a 2016 Toyota Mirai Fuel Cell (FC) vehicle based on chassis dynamometer testing. The 114.6 kW FC stack has a high dynamic response, which makes this powertrain a FC-dominant hybrid electric vehicle. The measured peak efficiency is 66.0% FC stack and 63.7% FC system with an idle hydrogen flow rate of 4.39 g/hr. The high FC system efficiencies at low loads match typical vehicle power spectrums, resulting in a high average vehicle efficiency of 62% compared to 45% and 23% for a hybrid electric vehicle and a conventional vehicle, respectively. An energy breakdown accounts for the FC stack losses, FC system losses, air compressor loads, and heater loads for different drive cycles and different thermal conditions. The cold-start North American city drive cycle (UDDS) energy consumption values are, respectively, 758, 581, 226, and 321 Wh/km at ambient conditions of -18 degrees C, -7 degrees C, -25 degrees C and 35 degrees C with 850 W/m 2 of solar loading. The FC system shutdown and startup processes at temperatures below the freezing point contribute to the increased hydrogen consumption. Additionally, the raw test data files are available for download, thus providing the research community with a public reference data on a modern production automotive FC system.
Antibody Fucosylation Lowers the FcγRIIIa/CD16a Affinity by Limiting the Conformations Sampled by the N162-Glycan
Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine(N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), reduces Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to afucosylated IgG1 Fc, however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan which impinges upon the linkage between the αmannose(1- 6)βmannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution NMR spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates antibody affinity indirectly through reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineering to enhance intermolecular glycan-glycan contacts will likely provide limited improvement and future designs should maximize affinity by maintaining CD16a Asn162-glycan conformational heterogeneity
New echocardiographic windows for quantitative determination of aortic regurgitation volume using color Doppler flow convergence and vena contracta
Color Doppler images of aortic regurgitation (AR) flow acceleration, flow convergence (FC), and the vena contracta (VC) have been reported to be useful for evaluating severity of AR. However, clinical application of these methods has been limited because of the difficulty in clearly imaging the FC and VC. This study aimed to explore new windows for imaging the FC and VC to evaluate AR volumes in patients and to validate this in animals with chronic AR. Forty patients with AR and 17 hemodynamic states in 4 sheep with strictly quantified AR volumes were evaluated. A Toshiba SSH 380A with a 3.75-MHz transducer was used to image the FC and VC. After routine echo Doppler imaging, patients were repositioned in the right lateral decubitus position, and the FC and VC were imaged from high right parasternal windows. In only 15 of the 40 patients was it possible to image clearly and measure accurately the FC and VC from conventional (left decubitus) apical or parasternal views. In contrast, 31 of 40 patients had clearly imaged FC regions and VCs using the new windows. In patients, AR volumes derived from the FC and VC methods combined with continuous velocity agreed well with each other (r = 0.97, mean difference = -7.9 ml +/- 9.9 ml/beat). In chronic animal model studies, AR volumes derived from both the VC and the FC agreed well with the electromagnetically derived AR volumes (r = 0.92, mean difference = -1.3 +/- 4.0 ml/beat). By imaging from high right parasternal windows in the right decubitus position, complementary use of the FC and VC methods can provide clinically valuable information about AR volumes.
Designed proteins for pH switchable antibody purification
Modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are provided. In some examples, a modified Fc-binding domain includes or consists of the amino acid sequence of any one of SEQ ID NOs: 4, 6, or 8. Nucleic acids encoding the modified Fc-binding domains or polypeptides including one or more of the modified Fc-binding domains are also provided. In some embodiments, the nucleic acids are included in a vector and may be operably linked to a promoter. Methods for purifying a polypeptide including one or more Fc regions are also provided. Kits and reagents including the modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are provided. In some examples, the modified Fc-binding domain polypeptides or polypeptides including one or more modified Fc-binding domains are linked to a solid support.
The influence of the spectral emissivity of flat-plate calibrators on the calibration of IR thermometers
Flat Calibrators (FC) are an option for calibration of infrared thermometers (IT) with a fixed large target. FCs are neither blackbodies, nor gray-bodies; their spectral emissivity is lower than one and depends on wavelength. Nevertheless they are used as gray-bodies with a nominal emissivity value. FCs can be calibrated radiometrically using as reference a calibrated IR thermometer (RT). If an FC will be used to calibrate ITs that work in the same spectral range as the RT then its calibration is straightforward: the actual FC spectral emissivity is not required. This result is valid for any given fixed emissivity assessed to the FC. On the other hand, when the RT working spectral range does not match with that of the ITs to be calibrated with the FC then it is required to know the FC spectral emissivity as part of the calibration process. For this purpose, at CENAM, we developed an experimental setup to measure spectral emissivity in the infrared spectral range, based on a Fourier transform infrared spectrometer. Not all laboratories have emissivity measurement capability in the appropriate wavelength and temperature ranges to obtain the spectral emissivity. Thus, we present an estimation of the error introduced when the spectral range of the RT used to calibrate an FC and the spectral ranges of the ITs to be calibrated with the FC do not match. Some examples are developed for the cases when RT and IT spectral ranges are [8,13] μm and [8,14] μm respectively.
Real-time three-dimensional color doppler evaluation of the flow convergence zone for quantification of mitral regurgitation: Validation experimental animal study and initial clinical experience
BACKGROUND: Pitfalls of the flow convergence (FC) method, including 2-dimensional imaging of the 3-dimensional (3D) geometry of the FC surface, can lead to erroneous quantification of mitral regurgitation (MR). This limitation may be mitigated by the use of real-time 3D color Doppler echocardiography (CE). Our objective was to validate a real-time 3D navigation method for MR quantification. METHODS: In 12 sheep with surgically induced chronic MR, 37 different hemodynamic conditions were studied with real-time 3DCE. Using real-time 3D navigation, the radius of the largest hemispherical FC zone was located and measured. MR volume was quantified according to the FC method after observing the shape of FC in 3D space. Aortic and mitral electromagnetic flow probes and meters were balanced against each other to determine reference MR volume. As an initial clinical application study, 22 patients with chronic MR were also studied with this real-time 3DCE-FC method. Left ventricular (LV) outflow tract automated cardiac flow measurement (Toshiba Corp, Tokyo, Japan) and real-time 3D LV stroke volume were used to quantify the reference MR volume (MR volume = 3DLV stroke volume - automated cardiac flow measurement). RESULTS: In the sheep model, a good correlation and agreement was seen between MR volume by real-time 3DCE and electromagnetic (y = 0.77x + 1.48, r = 0.87, P <.001, delta = -0.91 +/- 2.65 mL). In patients, real-time 3DCE-derived MR volume also showed a good correlation and agreement with the reference method (y = 0.89x - 0.38, r = 0.93, P <.001, delta = -4.8 +/- 7.6 mL). CONCLUSIONS: real-time 3DCE can capture the entire FC image, permitting geometrical recognition of the FC zone geometry and reliable MR quantification.
Purification and identification of the fusicoccin binding protein from oat root plasma membrane
Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.
Revisiting the definition of field capacity as a functional parameter in a layered agronomic soil profile beneath irrigated maize
The soil water content at the condition of field capacity (θ FC ) is a key parameter in irrigation scheduling and has been suggested to be determined by running a synthetic drainage experiment until the flux rate (q) at the bottom of the soil profile achieves a predefined negligible value (q FC ). We question the impact of q FC on the assessment of field capacity. Moreover, calculating θ FC as the integral mean of the water content profile when q is equal to q FC is strictly valid only for uniform soil profiles. By contrast, this practice is ambiguous and biased for stratified soil profiles due to the soil water content discontinuity at the layer interfaces. In this study, the concept of field capacity was revisited and adapted to practical agronomic heuristics. By resorting to the assessment of root-zone water storage capacity (W), we envision field capacity as a functional hydraulic parameter derived from synthetic irrigation scheduling scenarios to minimize drought stress, drainage, and nitrate leachate below the root zone. A functional analysis was carried out on a 135-cm-thick layered soil profile beneath maize in eastern Nebraska. On-farm irrigation scheduling applications and agricultural practices were recorded for 20 years (2001–2020) at a daily time step. Hydrus-1D was calibrated and validated with direct measurements of the soil water retention curve and soil water content data, respectively, in each soil layer. A set of functional field capacity values was derived from 24 irrigation scheduling scenarios, and the optimal water storage capacity at field capacity (W FC ) was approximately 50 cm (corresponding to about 80% saturation in the soil profile). An average irrigation amount of 217.5 mm distributed over 21 events was obtained by using optimal irrigation scheduling, which was initiated when the matric pressure head took on a value of –700 cm and the irrigation rate was set at 1.0 cm d –1 . This irrigation practice ensured water storage at approximately the same level (ideally at W FC ) by sustaining only evapotranspiration fluxes in the uppermost portion of the root zone and by limiting excessive drainage. This protocol can be transferred to other agricultural fields.
Engineered IL-18 variants with half-life extension and improved stability for cancer immunotherapy
Background The pro-inflammatory cytokine, interleukin-18 (IL-18), plays an instrumental role in bolstering anti-tumor immunity. However, the therapeutic application of IL-18 has been limited due to its susceptibility to neutralization by IL-18 binding protein (IL-18BP), short in vivo half-life, and unfavorable physicochemical properties. Methods In order to overcome the poor drug-like properties of IL-18, we installed an artificial disulfide bond, removed the native, unpaired cysteines, and fused the stabilized cytokine to an IgG Fc domain. The stability, potency, pharmacokinetic and pharmacodynamic properties as well as efficacy of disulfide-stabilized IL-18 Fc-fusion (dsIL-18-Fc) were assessed via in vitro and in vivo studies. Results The stability and mammalian host cell production yields of dsIL-18-Fc were improved, compared to the wild-type (WT) cytokine, while maintaining its biological potency and interactions with IL-18 receptor α (IL-18Rα) and IL-18BP. Recombinant fusion of the cytokine to an IgG Fc domain provided extended half-life. Notably, despite maintaining sensitivity to IL-18BP, dsIL-18-Fc was effective at activating both T and natural killer (NK) cells, and elicited a strong anti-tumor response, either as a single agent, or in conjunction with anti-programmed cell death-ligand 1 (anti-PD-L1) therapy. Conclusions We engineered IL-18 for reinforced stability, extended half-life, and improved manufacturability. The therapeutic benefit of dsIL-18-Fc, coupled with a more favorable manufacturability profile and enhanced drug-like properties, underscores the potential utility of this engineered cytokine in cancer immunotherapy.
Non-Additive Voltametric Currents From a Mixture of Two, Three and Four Redox-Active Compounds and Electroanalytical Implications
We have published recently the effect of dissimilar diffusion coefficients on the size of the voltammetric waves from a mixture of two redox-active compounds. Similarly, at the potential range where three redox-active species, decamethylferrocene (dMeFc), ferrocene (Fc) and N-methylphenothiazine (MePTZ), are oxidized simultaneously with rates controlled by linear diffusion, electrogenerated radicals diffusing outwards from the electrode react with the original species diffusing towards the electrode from the bulk; thus, Fc(+) reacts with dMeFc producing Fc and dMeFc(+), while MePTZ(+) reacts both with dMeFc producing MePTZ and dMeFc(+), and with Fc producing MePTZ and Fc(+). These reactions replace dMeFc with Fc at the second plateau, and both dMeFc and Fc with MePTZ at the third plateau. Since the diffusion coefficients of the three species are not equal, the mass-transfer limited currents of the second and the third oxidation wave plateaus change by approx. 10%. Numerical simulations of the experimental voltamograms support this mechanism. Similar results were also obtained for a mixture of four redoxactive compounds. The implications of this non-additive nature of currents on: (a) the use of internal voltammetric standards for quantitative analysis of a mixture of redox-active compounds; and, (b) the half wave potentials (E1/2) of the 2nd, 3rd and 4th waves for qualitative analysis, will be discussed.
Architectural Analysis of Complex Evolving Systems of Systems
The goal of this collaborative project between FC-MD, APL, and GSFC and supported by NASA IV&V Software Assurance Research Program (SARP), was to develop a tool, Dynamic SAVE, or Dyn-SAVE for short, for analyzing architectures of systems of systems. The project team was comprised of the principal investigator (PI) from FC-MD and four other FC-MD scientists (part time) and several FC-MD students (full time), as well as, two APL software architects (part time), and one NASA POC (part time). The PI and FC-MD scientists together with APL architects were responsible for requirements analysis, and for applying and evaluating the Dyn-SAVE tool and method. The PI and a group of FC-MD scientists were responsible for improving the method and conducting outreach activities, while another group of FC-MD scientists were responsible for development and improvement of the tool. Oversight and reporting was conducted by the PI and NASA POC. The project team produced many results including several prototypes of the Dyn-SAVE tool and method, several case studies documenting how the tool and method was applied to APL s software systems, and several published papers in highly respected conferences and journals. Dyn-SAVE as developed and enhanced throughout this research period, is a software tool intended for software developers and architects, software integration testers, and persons who need to analyze software systems from the point of view of how it communicates with other systems. Using the tool, the user specifies the planned communication behavior of the system modeled as a sequence diagram. The user then captures and imports the actual communication behavior of the system, which is then converted and visualized as a sequence diagram by Dyn-SAVE. After mapping the planned to the actual and specifying parameter and timing constraints, Dyn-SAVE detects and highlights deviations between the planned and the actual behavior. Requirements based on the need to analyze two inter-system communication protocols that are representative of protocols used in the Aerospace industry have been specified. The protocols are related: APL s Common Ground System (CGS) as used in the MErcury Surface, Space ENvironment, GEochemistry, and Ranging (MESSENGER) and the Radiation Belt Space Probes (RBSP) missions. The analyzed communications were implementations of the Telemetry protocol and the CCSDS File Delivery Protocol (CFDP) protocol. Based on these requirements, three prototypes of Dyn-SAVE were developed and applied to these protocols. The application of Dyn-SAVE to these protocols resulted in the detection of several issues. Dyn-SAVE was also applied to several Testbeds that have previously been used for experimentation earlier on this project, as well as, to other protocols and logs for testing its broader applicability. For example, Dyn-SAVE was used to analyze 1) the communication pattern between a web browser and a web server, 2) the system log of a computer in order to detect offnominal computer shut-down behavior, and 3) the actual test cases of NASA Goddard s Core Flight System (CFS) and automatically generated test cases in order to determine the overlap between the two sets of test cases. In all cases, Dyn-SAVE assisted in providing insightful conclusions about each of the cases identified above.
Electrolyte effects on the electrochemical performance of microemulsions
Here, we report a study of the electrochemistry of Ferrocene (Fc) in a Tween®20 (polyethylene glycol (20) sorbitan monolaurate) /1-butanol/ water/Toluene (TBWT) microemulsion system, focusing on the effects of electrolyte components. Resistance and conductivity measurements are used along with Cyclic Voltammetry (CV) to characterize the effects of supporting electrolyte, such as Bu 4 NBF 4 and KNO 3 , on the electrochemical performance and transport properties of Fc. With increasing Fc-containing oil phase concentration in the microemulsion, the observed peak current density fails to increase proportionately with the Fc concentration. Adding 0.05 m Bu 4 NBF 4 in the oil phase significantly increases the current density, presumably by lowering the resistance of microemulsion and promoting the accessibility of Fc in the system. Moreover, increasing the aqueous phase supporting electrolyte from 0.5 m KNO 3 to 1 m KNO 3 does not exhibit an analogous effect. We discuss these results in the context of our working hypothesis for microemulsion organization and its implications for electron transfer to Fc.