Replication-Based Rearrangements Are a Common Mechanism for SNCA Duplication in Parkinson's Disease.

BACKGROUND: SNCA multiplication is a genomic cause of familial PD, showing dosage-dependent toxicity. Until now, nonallelic homologous recombination was suggested as the mechanism of SNCA duplication, based on various types of repetitive elements found in the spanning region of the breakpoints. However, the sequence at the breakpoint was analyzed only for 1 case. OBJECTIVES: We have analyzed the breakpoint sequences of 6 patients with PD who had duplicated SNCA using whole-genome sequencing data to elucidate the mechanism of SNCA duplication. METHODS: Six patient samples with SNCA duplication underwent whole-genome sequencing. The duplicated regions were defined with nucleotide-resolution breakpoints, which were confirmed by junction polymerase chain reaction and Sanger sequencing. The search for potential non-B DNA-forming sequences and stem-loop structure predictions was conducted. RESULTS: Duplicated regions ranged from the smallest region of 718.3 kb to the largest one of 4,162 kb. Repetitive elements were found at 8 of the 12 breakpoint sequences on each side of the junction, but none of the pairs shared overt homologies. Five of these six junctions had microhomologies (2-4 bp) at the breakpoint, and a short stretch of sequences was inserted in 3 cases. All except one junction were located within or next to stem-loop structures. CONCLUSION: Our study has determined that homologous recombination mechanisms involving repetitive elements are not the main cause of the duplication of SNCA. The presence of microhomology at the junctions and their position within stem-loop structures suggest that replication-based rearrangements may be a common mechanism for SNCA amplification. © 2020 International Parkinson and Movement Disorder Society.

Exogenous neutrophil elastase enters bronchial epithelial cells and suppresses cigarette smoke extract-induced heme oxygenase-1 by cleaving sirtuin 1.

An imbalance between oxidative stress and antioxidant activity plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Cigarette smoke, a major risk factor of COPD, induces cellular oxidative stress, but levels of antioxidants such as heme oxygenase-1 (HO-1) are reduced in individuals with severe COPD. In this study, we evaluated the molecular mechanism of reduced HO-1 expression in human bronchial epithelial cells. We found that cigarette smoke extract (CSE) increases HO-1 levels via activation of NFE2-related factor 2 (Nrf2). However, pretreating cells with the protease neutrophil elastase (NE) suppressed the CSE-induced expression of HO-1 mRNA and protein. NE also decreased the sirtuin 1 (SIRT1) level, but did not inhibit CSE-induced nuclear translocation and DNA-binding activity of Nrf2. Transfection of cells with a Myc/His-tagged SIRT1 expression vector completely blocked the NE-mediated suppression of HO-1 expression. We further noted that the NE-induced down-regulation of SIRT1 was not due to decreased transcription or proteasomal/lysosomal degradation or loss of solubility. Immunofluorescence staining revealed that NE enters the cell cytoplasm, and we observed that NE directly cleaved SIRT1 in vitro, indicating that SIRT1 levels are decreased via direct degradation by internalized NE. Of note, we observed decreased SIRT1 levels in NE-treated primary human bronchial epithelial cells and in lung homogenates from both smokers and patients with COPD. In conclusion, NE suppresses CSE-induced HO-1 expression by cleaving SIRT1. This finding indicates the importance of cross-talk between oxidative stress and protease responses in the pathogenesis of COPD.

The effect of imidazolium cations on the structure and activity of the Candida antarctica Lipase B enzyme in ionic liquids.

In order to understand how cations affect the structural changes and enzyme activity of Lipase B from Candida antarctica, we performed all-atom molecular dynamics simulations of CALB in four types of ionic liquids (ILs) with varying sizes of imidazolium cations and correlated these results with the experimentally determined CALB activity. The imidazolium cations under study differ in the alkyl tail length in the following order: [Emim](+) < [Bmim](+) < [Hmim](+) < [Omim](+). We observed that the best enzyme activity and structural stability of CALB are obtained in [Bmim][TfO] and [Hmim][TfO]. In contrast, in [Emim][TfO], bonding of [TfO](-) to LYS-290 disrupts the interactions between LYS-290 and ILE-285, which leads to a closed catalytic gate conformation with low accessibility of substrates to the catalytic triad. In [Omim][TfO], strong hydrophobic interactions between [Omim](+) and LEU-278 result in a significant loss of the secondary structure of the α-10 helix and cause the exposure of the catalytic triad to ILs, which affects the stability of the catalytic triad and consequently deteriorates the enzyme activity. Overall, our study indicates that a high ion coordination number ([Emim][TfO]) or the presence of a long hydrophobic tail ([Omim][TfO]) can facilitate ion-protein interactions that cause structural distortions and a decrease in CALB enzyme activity in ILs.

The relationship between enhanced enzyme activity and structural dynamics in ionic liquids: a combined computational and experimental study.

Candida antarctica lipase B (CALB) is an efficient biocatalyst for hydrolysis, esterification, and polymerization reactions. In order to understand how to control enzyme activity and stability we performed a combined experimental and molecular dynamics simulation study of CALB in organic solvents and ionic liquids (ILs). Our results demonstrate that the conformational changes of the active site cavity are directly related to enzyme activity and decrease in the following order: [Bmim][TfO] > tert-butanol > [Bmim][Cl]. The entrance to the cavity is modulated by two isoleucines, ILE-189 and ILE-285, one of which is located on the α-10 helix. The α-10 helix can substantially change its conformation due to specific interactions with solvent molecules. This change is acutely evident in [Bmim][Cl] where interactions of LYS-290 with chlorine anions caused a conformational switch between α-helix and turn. Disruption of the α-10 helix structure results in a narrow cavity entrance and, thus, reduced the activity of CALB in [Bmim][Cl]. Finally, our results show that the electrostatic energy between solvents in this study and CALB is correlated with the structural changes leading to differences in enzyme activity.

Optimization of lipase-catalyzed synthesis of caffeic acid phenethyl ester in ionic liquids by response surface methodology.

Lipase-catalyzed caffeic acid phenethyl ester (CAPE) synthesis in ionic liquid, 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]), was investigated in this study. The effects of several reaction conditions, including reaction time, reaction temperature, substrate molar ratio of phenethyl alcohol to caffeic acid (CA), and weight ratio of enzyme to CA, on CAPE yield were examined. In a single parameter study, the highest CAPE yield in [Emim][Tf(2)N] was obtained at 70 °C with a substrate molar ratio of 30:1 and weight ratio of enzyme to CA of 15:1. Based on these results, response surface methodology (RSM) with a 3-level-4-factor central composite rotatable design (CCRD) was adopted to evaluate enzymatic synthesis of CAPE in [Emim][Tf(2)N]. The four major factors were reaction time (36-60 h), reaction temperature (65-75 °C), substrate molar ratio of phenethyl alcohol to CA (20:1-40:1), and weight ratio of enzyme to CA (10:1-20:1). A quadratic equation model was used to analyze the experimental data at a 95 % confidence level (p < 0.05). A maximum conversion yield of 99.8 % was obtained under the optimized reaction conditions [60 h, 73.7 °C, substrate molar ratio of phenethyl alcohol to CA (27.1:1), and weight ratio of enzyme to CA (17.8:1)] established by our statistical method, whereas the experimental conversion yield was 96.6 ± 2 %.

Effect of ionic liquids on enzymatic synthesis of caffeic acid phenethyl ester.

Although caffeic acid phenethyl ester (CAPE), an active flavonoid, plays an important role in the antioxidant activity of honeybee propolis, the isolation of CAPE from honeybee propolis is time-consuming due to wide variety of impurities present. Therefore, biochemical method to synthesize CAPE was investigated in this study. Since ionic liquids (ILs) possess some unique characteristics as appreciated alternatives to conventional solvents for certain biotransformation, the effect of ILs as reaction media for enzymatic synthesis of CAPE was assessed. Several factors including substrate molar ratio, and reaction temperature affecting the conversion yield of lipase-catalyzed CAPE synthesis were also investigated. Reaction yields were significantly higher in hydrophobic ILs than in hydrophilic ILs (almost zero). Among nine hydrophobic ILs tested, the highest conversion of synthetic reaction was obtained in 1-ethyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide ([Emim][Tf(2)N]). A reaction temperature of 70 °C was found to give high conversion. In addition, optimal substrate molar ratio between phenethyl alcohol and caffeic acid (CA) was decreased significantly from 92:1 to 30:1 when ILs were used instead of isooctane.

PTX Instructs the Development of Lung-Resident Memory T Cells in Bordetella pertussis Infected Mice.

Whooping cough is a severe, highly contagious disease of the human respiratory tract, caused by Bordetellapertussis. The pathogenicity requires several virulence factors, including pertussis toxin (PTX), a key component of current available vaccines. Current vaccines do not induce mucosal immunity. Tissue-resident memory T cells (Trm) are among the first lines of defense against invading pathogens and are involved in long-term protection. However, the factors involved in Trm establishment remain unknown. Comparing two B.pertussis strains expressing PTX (WT) or not (ΔPTX), we show that the toxin is required to generate both lung CD4+ and CD8+ Trm. Co-administering purified PTX with ΔPTX is sufficient to generate these Trm subsets. Importantly, adoptive transfer of lung CD4+ or CD8+ Trm conferred protection against B. pertussis in naïve mice. Taken together, our data demonstrate for the first time a critical role for PTX in the induction of mucosal long-term protection against B. pertussis.

An enhancer mutant of Arabidopsis salt overly sensitive 3 mediates both ion homeostasis and the oxidative stress response.

The myristoylated calcium sensor SOS3 and its interacting protein kinase, SOS2, play critical regulatory roles in salt tolerance. Mutations in either of these proteins render Arabidopsis thaliana plants hypersensitive to salt stress. We report here the isolation and characterization of a mutant called enh1-1 that enhances the salt sensitivity of sos3-1 and also causes increased salt sensitivity by itself. ENH1 encodes a chloroplast-localized protein with a PDZ domain at the N-terminal region and a rubredoxin domain in the C-terminal part. Rubredoxins are known to be involved in the reduction of superoxide in some anaerobic bacteria. The enh1-1 mutation causes enhanced accumulation of reactive oxygen species (ROS), particularly under salt stress. ROS also accumulate to higher levels in sos2-1 but not in sos3-1 mutants. The enh1-1 mutation does not enhance sos2-1 phenotypes. Also, enh1-1 and sos2-1 mutants, but not sos3-1 mutants, show increased sensitivity to oxidative stress. These results indicate that ENH1 functions in the detoxification of reactive oxygen species resulting from salt stress by participating in a new salt tolerance pathway that may involve SOS2 but not SOS3.

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids.

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf(2)N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf(2)N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.

Beta-glucosidase coating on polymer nanofibers for improved cellulosic ethanol production.

Beta-glucosidase (betaG) can relieve the product inhibition of cellobiose in the cellulosic ethanol production by converting cellobiose into glucose. For the potential recycled uses, betaG was immobilized and stabilized in the form of enzyme coating on polymer nanofibers. The betaG coating (EC-betaG) was fabricated by crosslinking additional betaG molecules onto covalently attached betaG molecules (CA-betaG) via glutaraldehyde treatment. The initial activity of EC-betaG was 36 times higher than that of CA-betaG. After 20 days of incubation under shaking, CA-betaG and EC-betaG retained 33 and 91% of each initial activity, respectively. Magnetic nanofibers were also used for easy recovery and recycled uses of betaG coating. It is anticipated that the recycled uses of highly active and stable betaG coating can improve the economics of cellulosic ethanol production so long as economical materials are employed as a host of enzyme immobilization.

Highly stable trypsin-aggregate coatings on polymer nanofibers for repeated protein digestion.

A stable and robust trypsin-based biocatalytic system was developed and demonstrated for proteomic applications. The system utilizes polymer nanofibers coated with trypsin aggregates for immobilized protease digestions. After covalently attaching an initial layer of trypsin to the polymer nanofibers, highly concentrated trypsin molecules are crosslinked to the layered trypsin by way of a glutaraldehyde treatment. This process produced a 300-fold increase in trypsin activity compared with a conventional method for covalent trypsin immobilization, and proved to be robust in that it still maintained a high level of activity after a year of repeated recycling. This highly stable form of immobilized trypsin was resistant to autolysis, enabling repeated digestions of BSA over 40 days and successful peptide identification by LC-MS/MS. This active and stable form of immobilized trypsin was successfully employed in the digestion of yeast proteome extract with high reproducibility and within shorter time than conventional protein digestion using solution phase trypsin. Finally, the immobilized trypsin was resistant to proteolysis when exposed to other enzymes (i.e., chymotrypsin), which makes it suitable for use in "real-world" proteomic applications. Overall, the biocatalytic nanofibers with trypsin aggregate coatings proved to be an effective approach for repeated and automated protein digestion in proteomic analyses.

Separation of D-psicose and D-fructose using simulated moving bed chromatography.

Simulated moving bed (SMB) processes have been widely used in the sugar industries with ion-exchange resin as a stationary phase. D-psicose, a rare monosaccharide known as a valuable pharmaceutical substrate, was synthesized by the enzymatic conversion from D-fructose. The SMB process was adopted to separate D-psicose from D-fructose. Before the SMB experiment, the reaction mixture including D-psicose and D-fructose was treated by a deashing process to remove contaminants, such as buffers, proteins, and other organic materials. Four columns packed with Dowex 50WX4-Ca2+ (200-400 mesh) ion-exchange resins were used in the four-zone SMB. Single-step frontal analysis was performed to estimate the isotherm parameters of each monosaccharide. The operating conditions of the SMB process were determined based on the Equilibrium Theory. According to the simulation of the SMB process, the purity and yield of extract product (D-psicose) achieved were 99.04 and 97.46%, respectively and those of raffinate product (D-fructose) were 99.06 and 99.53%, respectively. Under the optimized operating condition, complete separation (extract purity = 99.36%, raffinate purity = 99.67%) was achieved experimentally.

Toxicity studies of cremophor-free paclitaxel solid dispersion formulated by a supercritical antisolvent process.

To evaluate the acute toxicity of a paclitaxel solid dispersion formulation, single dose studies in ICR mice were carried out for injectable excipients, paclitaxel solid dispersion powder, and Taxol. In the dose range of excipients used for preparing paclitaxel solid dispersion, each excipient was clinically safe, and the LD(50) for exicipients was higher than 2,000 mg/kg for both males and females. In this study, there were no remarkable clinical signs or deaths related to paclitaxel solid dispersion even at doses up to 160 mg/kg of paclitaxel. But Taxol resulted in clinical signs when it contained more than 30 mg/mL paclitaxel. The LD(50) for paclitaxel solid dispersion was above 160 mg/kg and the LD(50) for Taxol was 31.3 mg/kg, more than 5 times lower than that of paclitaxel solid dispersion. However, paclitaxel solid dispersion could not be administered i.v. at a dose exceeding 160 mg/kg, because of high viscosity. To evaluate the nephrotoxicity of paclitaxel solid dispersion, plasma level of creatinine and kidney weight were measured and compared to Taxol. At the doses administered, paclitaxel solid dispersion did not change creatinine clearance, while Taxol killed all animals at doses >15 mg/kg. To investigate membrane damage when paclitaxel formulations were injected, hemolytic activity was determined for different concentrations. Paclitaxel solid dispersion showed about 10% hemolytic activity, whereas Taxol showed about 40% hemolytic activity when it contained 2 mg of paclitaxel. Comparisons with the LD(50) value, nephrotoxicity, and hemolytic activity of Taxol suggested that Cremophor-free paclitaxel solid dispersion as an injectable formulation is a promising approach to increasing the safety and clinical efficacy of paclitaxel for treatment of cancer.

Development of novel ibuprofen-loaded solid dispersion with improved bioavailability using aqueous solution.

To develop a novel ibuprofen-loaded solid dispersion with enhanced bioavailability, various ibuprofen-loaded solid dispersions were prepared with water, HPMC and poloxamer. The effect of HPMC and poloxamer on aqueous solubility of ibuprofen was investigated. The dissolution and bioavailability of solid dispersion in rats were then evaluated compared to ibuprofen powder. When the amount of carrier increased with a decreased in HPMC/poloxamer ratio, the aqueous solubility of ibuprofen was elevated. The solid dispersion composed of ibuprofen/HPMC/poloxamer at the weight ratio of 10:3:2 improved the drug solubility approximately 4 fold. It gave significantly higher initial plasma concentration, AUC and Cmax of drug than did ibuprofen powder in rats. The solid dispersion improved the bioavailability of drug about 4-fold compared to ibuprofen powder. Thus, this ibuprofen-loaded solid dispersion with water, HPMC and poloxamer was a more effective oral dosage form for improving the bioavailability of poor water-soluble ibuprofen.

Whole-Cell Biocatalysis in Ionic Liquids.

The use of whole-cell biocatalysis in ionic liquid (IL)-containing systems has attracted increasing attention in recent years. Compared to bioreactions catalyzed by isolated enzymes, the major advantage of using whole cells in biocatalytic processes is that the cells provide a natural intracellular environment for the enzymes to function with in situ cofactor regeneration. To date, the applications of whole-cell biocatalysis in IL-containing systems have focused on the production of valuable compounds, mainly through reduction, oxidation, hydrolysis, and transesterification reactions. The interaction mechanisms between the ILs and biocatalysts in whole-cell biocatalysis offer the possibility to effectively integrate ILs with biotransformation. This chapter discusses these interaction mechanisms between ILs and whole-cell catalysts. In addition, examples of whole-cell catalyzed reactions with ILs will also be discussed. Graphical Abstract.

Separation characteristics of hydrophilic ionic liquids from ionic liquids-water solution by ultrasonic atomization.

Ionic liquids (ILs) have attracted much attention as promising alternatives for volatile organic solvents. Although the applications of ILs have been found in a diverse range of fields, there are a limited number of methods for the recovery of ILs so far. As an efficient separation method, therefore, ultrasonic atomization has been attempted to recover hydrophilic ILs, [Bmim][BF4], from ILs-water solution. In order to examine the separation characteristics of hydrophilic ILs-water solution, ultrasonic atomization of hydrophilic ILs-water solution was performed under various operating conditions such as initial ILs concentration, ultrasonic electric power, carrier gas flow rate, and operating temperature. The result showed that hydrophilic ILs recovery yield increased with a decrease in ultrasonic electric power, gas velocity, and temperature. As an increase in initial ILs concentration, however, higher ILs recovery yield was obtained. After 6 h of ultrasonic atomization of 50% (v/v) [Bmim][BF4]-water solution, 93.4% of initial ILs amount was recovered without any changes in their structure at ultrasonic power of 10 W, carrier gas flow of 5 L/min and temperature of 20 °C. It demonstrated that ultrasonic atomization could be used for the recovery of ILs from ILs-aqueous solution.

Enhanced lipase-catalyzed synthesis of sugar fatty acid esters using supersaturated sugar solution in ionic liquids.

A solvent-mediated method (SMM) was used to prepare supersaturated sugar solutions in hydrophobic and mixture of hydrophilic/hydrophobic ionic liquids (ILs), namely, [Bmim][Tf2N] and [Bmim][TfO]/[Bmim][Tf2N], respectively. In this method, sugars were first solubilized in a mixture of organic solvent and water (i.e. methanol:water, 1:1 v/v), and then added to [Bmim][Tf2N] and/or [Bmim][TfO]/[Bmim][Tf2N] mixture. Supersaturated sugar solution in ILs were obtained by removing organic solvents and water under vacuum evaporation. Sugar solubilities in ILs, especially in hydrophobic IL ([Bmim][Tf2N]) and in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM were greater than in ILs prepared using water-mediated method (WMM), which suggested methanol aided sugar solvation in hydrophobic media. In addition, interactions between glucose molecules and between glucose and methanol, water, and IL were investigated by all-atom molecular dynamics (MD) simulation. The MD simulation results showed that initial water and water/methanol molecules around glucose were gradually replaced by IL anions. Notably, SMM resulted in stronger interaction between IL anions and glucose than WMM, which was attributed to greater solubility of sugar in ILs prepared by SMM. Resultantly, the productivity of lipase-catalyzed production of glucose laurate using supersaturated glucose solution in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM was at least 1.76-fold greater than that obtained in IL mixture prepared by WMM.

Simple synthesis of functionalized superparamagnetic magnetite/silica core/shell nanoparticles and their application as magnetically separable high-performance biocatalysts.

Uniformly sized silica-coated magnetic nanoparticles (magnetite@silica) are synthesized in a simple one-pot process using reverse micelles as nanoreactors. The core diameter of the magnetic nanoparticles is easily controlled by adjusting the w value ([polar solvent]/[surfactant]) in the reverse-micelle solution, and the thickness of the silica shell is easily controlled by varying the amount of tetraethyl orthosilicate added after the synthesis of the magnetite cores. Several grams of monodisperse magnetite@silica nanoparticles can be synthesized without going through any size-selection process. When crosslinked enzyme molecules form clusters on the surfaces of the magnetite@silica nanoparticles, the resulting hybrid composites are magnetically separable, highly active, and stable under harsh shaking conditions for more than 15 days. Conversely, covalently attached enzymes on the surface of the magnetite@silica nanoparticles are deactivated under the same conditions.

Lipase-catalyzed synthesis of fatty acid sugar ester using extremely supersaturated sugar solution in ionic liquids.

The low solubility of sugars has hampered the lipase-catalyzed synthesis of fatty acid sugar esters in organic solvents and ionic liquids (ILs), because several solvents that are able to effectively dissolve sugars are detrimental to enzymes. In this work, in order to prepare a high concentration of sugars in ILs, we have developed a new procedure that entails mixing an aqueous sugar solution into ILs followed by removal of the water from the solution. The glucose concentrations in the supersaturated [Emim][TfO] and [Bmim][TfO] were 19 and 10 times higher, respectively, than the solubilities (6.1 and 4.8 g/L) of glucose in the ILs at 25 degrees C. Furthermore, the supersaturated glucose solutions in ILs were maintained over a long period of time without any significant loss of glucose. In ILs that were extremely supersaturated with glucose, lipase-catalyzed esterifications of glucose with vinyl laurate, and lauric acid were successfully carried out. The conversion increased from 8% to 96% at 1 day of reaction by using supersaturated solution in [Bmim][TfO] which had dissolved glucose concentration of 400% higher than its solubility, compared with the reaction using saturated glucose solution. By making the glucose concentration in ILs much higher than the solubility through our novel and simple method, the initial rate and conversion of the lipase-catalyzed reaction were significantly improved.