Cell-Free Synthesis of Selenoproteins in High Yield and Purity for Selective Protein Tagging

The selenol group of selenocysteine is much more nucleophilic than the thiol group of cysteine. Selenocysteine residues in proteins thus offer reactive points for rapid post-translational modification. Herein, we show that selenoproteins can be expressed in high yield and purity by cell-free protein synthesis by global substitution of cysteine by selenocysteine. Complete alkylation of solvent-exposed selenocysteine residues was achieved in 10 minutes with 4-chloromethylene dipicolinic acid (4Cl-MDPA) under conditions that left cysteine residues unchanged even after overnight incubation. Gd^III−Gd^III distances measured by double electron–electron resonance (DEER) experiments of maltose binding protein (MBP) containing two selenocysteine residues tagged with 4Cl-MDPA-Gd^III were indistinguishable from Gd^III−Gd^III distances measured of MBP containing cysteine reacted with 4Br-MDPA tags.     

Analysis of splenic and thymic lymphocyte subpopulations in chickens infected with Salmonella enteritidis

Lymphocytes expressing CD3, CD4, CD8, pan lymphocyte, IgA, IgG and IgM cell surface antigens were assessed by in the spleen and thymus of chickens following infection with Salmonella enteritidis using flow cytometric analysis. At 6 days post primary infection and 2 days post secondary infection with S. enteritidis, the percentages of IgA+ and IgM+ lymphocytes in the spleen were significantly increased (P < 0.05). At 2 days post secondary infection with S. enteritidis, the percentage of CD4+ T lymphocyte in the spleen and CD8+ T lymphocyte percentage in the thymus were significantly increased (P < 0.05). These results indicate that S. enteritidis infection induces changes in the spleen and thymus that reflect the dynamics of the host protective immune response.     

Spectral characterization of Dictyostelium autofluorescence

Dictyostelium discoideum is used extensively as a model organism for the study of chemotaxis. In recent years, an increasing number of studies of Dictyostelium chemotaxis have made use of fluorescence-based techniques. One of the major factors that can interfere with the application of these techniques in cells is the cellular autofluorescence. In this study, the spectral properties of Dictyostelium autofluorescence have been characterized using fluorescence microscopy. Whole cell autofluorescence spectra obtained using spectral imaging microscopy show that Dictyostelium autofluorescence covers a wavelength range from approximately 500 to 650 nm with a maximum at approximately 510 nm, and thus, potentially interferes with measurements of green fluorescent protein (GFP) fusion proteins with fluorescence microscopy techniques. Further characterization of the spatial distribution, intensity, and brightness of the autofluorescence was performed with fluorescence confocal microscopy and fluorescence fluctuation spectroscopy (FFS). The autofluorescence in both chemotaxing and nonchemotaxing cells is localized in discrete areas. The high intensity seen in cells incubated in the growth medium HG5 reduces by around 50% when incubated in buffer, and can be further reduced by around 85% by photobleaching cells for 5-7 s. The average intensity and spatial distribution of the autofluorescence do not change with long incubations in the buffer. The cellular autofluorescence has a seven times lower molecular brightness than eGFP. The influence of autofluorescence in FFS measurements can be minimized by incubating cells in buffer during the measurements, pre-bleaching, and making use of low excitation intensities. The results obtained in this study thus offer guidelines to the design of future fluorescence studies of Dictyostelium.     

Effect of Solvent,Preheating Temperature,and Time on the Ultrasonic Extraction of Phenolic Compounds from Cold‐Pressed Hempseed Cake

The effect of different solvents (aqueous methanol [70%, v/v], aqueous acetone [80%, v/v], and a solvent mixture [MA] of aqueous methanol [70%, v/v] and aqueous acetone [70%, v/v] in a ratio of 1:1 [v/v]), preheating temperatures (140, 160, and 180°C), and times of exposure (5, 15, and 30 min) on the ultrasonic extraction of the main phenolic compounds from hempseed cake (Cannabis sativa) was investigated. A simplified new high‐performance liquid chromatography (HPLC) method was developed to identify and quantify the main phenolics (namely, N‐trans‐caffeoyltyramine and cannabisin B) in the extracts. Two other main compounds, numbered 3 and 4 , were also detected. The results showed that the nature of the extracting solvent had a significant (P < 0.05) impact on the ultrasonic extraction of phenolic compounds. The acetone extracts exhibited the highest total phenolic content (TPC), followed by MA and methanol. The preheating temperature and time of exposure enhanced the TPC for all solvents examined. The main phenolics, N‐trans‐caffeoyltyramine, cannabisin B, and compound 3 , were positively affected by the temperature and time of exposure, irrespective of the solvents used. In sharp contrast, compound 4 appeared to be thermally sensitive: increasing preheating time and temperature decreased the yields of this compound. This study demonstrated that acetone was the most effective extracting solvent and that preheating enhanced the yield of the main phenolics.     

Optimisation of the number of IGBT devices in a series-parallel string

High-power semiconductor switches can be realised by connecting existing devices in series and parallel. The number of devices in series depends on the operating voltage of an application and the individual device voltage rating. For a given application, the use of higher voltage rated IGBTs leads to a fewer number of devices and vice versa. The total power loss of the series string equals to the sum of individual IGBT power losses and total loss increases with the increase in operating frequency. The level of increase in power loss depends on the device characteristics. For high current operation, the minimum number of devices depends on the current rating of individual device. In this paper, series IGBT string of six 1.2 kV, four 1.7 kV, two 3.3 kV and a single 6.5 kV IGBTs are simulated for a 4.5 kV/100 A application and power losses are analysed for different frequencies and duty cycles. This power loss analysis is extended for commercial IGBTs to compare the simulation results. The number of devices for minimum power loss depends on operating frequencies and power savings are significant both at low and high frequencies. In addition to the power losses, the other important issues in optimising the number of IGBTs are described in this paper. When IGBT modules are connected in parallel the principle of derating is applied to obtained reliable operation. This is explained with some examples.     

Attenuation of sinapic acid and sinapine-derived flavor-active compounds using a factorial-based pressurized high-temperature processing

De-oiled canola meals are sources of protein-containing flavor-active phenolic compounds. Conventional canola oil processing utilizes an excess amount of solvents and is associated with the release of high-intensity bitter flavor-active phenolic compounds, limiting the use of the canola meal. Recent advances in the extraction and isolation of the bitter favor-active phenolic compounds from canola by-products produce protein isolates, however, would benefit the industry by producing a side-stream ingredient rich in phenolics. High temperature and pressure-aided processing, namely the accelerated solvent extraction (ASE) was investigated to extract the flavor-active bitter molecules from the canola meal. The extractability of flavor-active phenolic compounds including the major sinapates, kaempferol derivatives, and other thermo-generative compounds including thomasidioc acid (TA) was evaluated. The effects of temperature, solvent extractant and concentration, and the particle size of the meal were examined on the extraction efficiency of these phenolic compounds. Extraction temperature (180°C) was the primary determinant (p < 0.05) for the attenuation of major sinapates including sinapine and sinapic acid. Both ethanol and methanol extractants at a concentration of 70% (v/v) significantly (p < 0.05) extracted the flavor-active phenolic compounds. The pressurized high temperature through optimized ASE conditions attenuated the bitter undesirable flavor-active phenolic molecules from canola meal, thereby facilitating a potential value-added phenolic-rich by-product.     

A simple robust method for synthesis of metallic copper nanoparticles of high antibacterial potency against E. coli

A method for preparation of copper nanoparticles (Cu-NPs) was developed by simple reduction of CuCl(2) in the presence of gelatin as a stabilizer and without applying stringent conditions like purging with nitrogen. The NPs were characterized by spectrophotometry, dynamic light scattering, x-ray diffraction, transmission electron microscopy, atomic force microscopy and x-ray photoelectron spectroscopy. The particles were about 50-60?nm in size and highly stable. The antibacterial activity of this Cu-NP on Gram-negative Escherichia coli was demonstrated by the methods of agar plating, flow cytometry and phase contrast microscopy. The minimum inhibitory concentration (3.0?μg?ml(-1)), minimum bactericidal concentration (7.5?μg?ml(-1)) and susceptibility constant (0.92) showed that this Cu-NP is highly effective against E. coli at a much lower concentration than that reported previously. Treatment with Cu-NPs made E. coli cells filamentous. The higher the concentration of Cu-NPs, the greater the population of filamentous cells; average filament size varied from 7 to 20?μm compared to the normal cell size of ～2.5?μm. Both filamentation and killing of cells by Cu-NPs (7.5 μg?ml(-1)) also occurred in an E. coli strain resistant to multiple antibiotics. Moreover, an antibacterial effect of Cu-NPs was also observed in Gram-positive Bacillus subtilis and Staphylococcus aureus, for which the values of minimum inhibitory concentration and minimum bactericidal concentration were close to that for E. coli.