Hierarchical superstructure aerogels for in situ biofluid metabolomics.

High-throughput biofluid metabolomics analysis for screening life-threatening diseases is urgently needed. However, the high salt content of biofluid samples, which introduces severe interference, can greatly limit the analysis throughput. Here, a new 3-D interconnected hierarchical superstructure, namely a "plasmonic gold-on-silica (Au/SiO2) double-layered aerogel", integrating distinctive features of an upper plasmonic gold aerogel with a lower inert silica aerogel was successfully developed to achieve in situ separation and storage of inorganic salts in the silica aerogel, parallel enrichment of metabolites on the surface of the functionalized gold aerogel, and direct desorption/ionization of enriched metabolites by the photo-excited gold aerogel for rapid, sensitive, and comprehensive metabolomics analysis of human serum/urine samples. By integrating all these unique advantages into the hierarchical aerogel, multifunctional properties were introduced in the SALDI substrate to enable its effective utilization in clinical metabolomics for the discovery of reliable metabolic biomarkers to achieve unambiguous differentiation of early and advanced-stage lung cancer patients from healthy individuals. This study provides insight into the design and application of superstructured nanomaterials for in situ separation, storage, and photoexcitation of multi-components in complex biofluid samples for sensitive analysis.

Use of T-Scan III in analyzing occlusal changes in molar fixed denture placement.

BACKGROUND: This study aims to analyze the longitudinal variation of occlusal force distribution prior to and after fixed restoration for molar full-crowns with T-SCAN III which provide reference for occlusal adjustment and long-term maintenance. METHODS: We enrolled a total of 20 patients who received conventional restorative treatment for molars. The occlusion examination was conducted in 3 stages (before placement, immediately after placement, and 3 months after placement) using T-SCAN III (Tekscan South Boston, MA, USA, 10.0) to examine and measure the occlusal contact areas of the full dentition. RESULTS: The results indicated that the occlusal force distribution in the molar region of the patients changed before and after the fixed restoration, but the percentages of occlusal force in the dental arch of the molar did not differ significantly before and after the restoration (P > 0.05). Three months after the fixed restoration, the percentage of occlusal force in the restored dental arches of lateral teeth increased significantly (P < 0.05). CONCLUSION: The results of this study indicated that the occlusal forces of the patients changed with tooth movement and adaptation, which is mainly reflected in the increasing occlusal force. Quantitative occlusal force analysis using T-SCAN III occlusal analyzer can provide more objective and accurate data to effectively guide clinical occlusion adjustments.

µ-PESI-based MS profiling combined with untargeted metabolomics analysis for rapid identification of red wine geographical origin.

BACKGROUND: The commercial value of red wine is strongly linked to its geographical origin. Given the large global market, there is great demand for high-throughput screening methods to authenticate the geographical source of red wine. However, only limited techniques have been established up to now. RESULTS: Herein, a sensitive and robust method, namely probe electrospray ionization mass spectrometry (µ-PESI-MS), was established to achieve rapid analysis at approximately 1.2 min per sample without any pretreatment. A scotch near the needle tip provides a fixed micro-volume for each analysis to achieve satisfactory ion signal reproducibility (RSD < 26.7%). In combination with a machine learning algorithm, 16 characteristic ions were discovered from thousands of detected ions and were utilized for differentiating red wine origin. Among them, the relative abundances of two characteristic metabolites (trigonelline and proline) correlated with geographical conditions (sun exposure and water stress) were identified, providing the rationale for differentiation of the geographical origin. CONCLUSION: The proposed µ-PESI-MS-based method demonstrates a promising high-throughput determination capability in red wine traceability.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study.

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 â„ƒ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (Ki) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between α4 and ß5 (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be - 46.1 ± 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148riceXIP-E98AnXyl11B, K143riceXIP-D138AnXyl11B and R148riceXIP-E189AnXyl11B provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148riceXIP@ 2HH1-E89AnXylB11@OE2 and N142riceXIP@ 1HD2-D138AnXylB11@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

Exploring competitive inhibition of a family 10 xylanase derived from Hu sheep rumen microbiota by Oryza sativa xylanase inhibitor protein: In vitro and in silico perspectives.

The catalytic domain of family GH10 xylanase, XYN-LXY_CD derived from Hu sheep rumen microbiota was expressed in Pichia pastoris X33. The special activity of reXYN-LXY_CD in the culture supernatant was 232.56 U/mg. The optima of reXYN-LXY_CD were 53 °C and pH 7.0. Recombinant Oryza sativa xylanase inhibitor protein (rePOsXIP) competitively inhibited reXYN-LXY_CD with an inhibition constant (Ki) value of 237.37 nM. The concentration of hydrolysates released from beechwood xylan by reXYN-LXY_CD reduced when rePOsXIP was added into the hydrolytic system. Fluorescence of reXYN-LXY_CD was statically quenched by rePOsXIP in a dose-dependent manner. The details in intermolecular interaction between XYN-LXY_CD and OsXIP were investigated by using molecular dynamics (MD) simulations, binding free energy computation and non-covalent interactions (NCI) analysis. Hydrogen bonding and van der Waals played indispensable roles in the XYN-LXY_CD/OsXIP interaction. The α-7 helix of OsXIP tightly occupied the catalytic pocket of XYN-LXY_CD with hydrogen bonding such as K239OsXIP-N261/Q292/E197XYN-LXY_CD (E197, the acid-base catalytic residue), D236OsXIP-K327XYN-LXY_CD and Q242OsXIP-E211/Q212XYN-LXY_CD. Based on the quantum theory of atoms in molecules (QTAIM), the Laplacian of electron density and core-valence bifurcation index of HZ3K239-OE2E197 were 0.1025 a.u. and 0.002218, respectively. Elucidating the mechanism underlying xylanase-inhibitor interactions might help construct XYN-LXY_CD mutants that gain resistance to XIPs and high catalytic activity, which would be more efficient in feed additives in livestock.

Pharmacokinetics and Bioequivalence of Misoprostol Tablets: An Open-Label, Randomized, Single-dose, Crossover Study With Healthy Chinese Volunteers.

Misoprostol is a synthetic prostaglandin E1 derivative that has been used to treat duodenal and gastric ulcers, and to prevent ulcers caused by nonsteroidal anti-inflammatory drugs in many countries. Misoprostol can also be used for medical abortion. This study aimed to investigate the pharmacokinetic profiles of misoprostol tablets (test product) by comparing them with Cytotec (200 µg) (reference product). To assess the bioequivalence between test and reference products, a two-sequence, two-period crossover study was conducted with 48 healthy Chinese subjects enrolled under fasting conditions. A validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was used to determine the concentration of misoprostol acid in plasma. A mixed model analysis of variance was used to calculate the bioequivalence of pharmacokinetic (PK) parameters. The point estimate of geometric mean ratios with 90% confidence intervals for the maximum observed concentration (Cmax ) and the area under the concentration-time curve (AUC0-t ) for misoprostol acid in reference and test products were 107.8% and 106.5%, respectively (range 80%-125%). Additionally, none of the secondary PK parameters presented significant differences. No severe or more than moderate adverse events were detected in the 48 subjects. However, one subject discontinued the treatment due to drug-related gastrointestinal reactions. All adverse events were mild with rates of 19.2% and 22.9% after the administration test and reference products, respectively. Overall, the bioequivalence between the two misoprostol products was demonstrated in fasting conditions, and all subjects tolerated both treatments.

Sensitivity of family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the T33I mutant to Oryza sativa xylanase inhibitor protein (OsXIP): An experimental and computational study.

Glycoside hydrolase (GH) family 10 and 11 xylanases are inhibited by many xylanase inhibitor proteins (XIPs). We recombinantly expressed the Oryza sativa xylanase inhibitor protein (OsXIP) in Pichia pastoris GS115, with a molecular mass of 47.0 kDa. Family GH11 Bacillus amyloliquefaciens xylanase A (BaxA) and the mutant T33I (DS199) were inhibited by the recombinant OsXIP (rePOsXIP) through competive inhibition, with corresponding inhibition constants (Ki) of 54.09 and 12.16 nM. After incubation with rePOsXIP (70 nM) at 40 °C for 40 min, inhibitory rates of reBaxA and DS199 (0.2 U) were 23.7% and 76.7%, respectively. Xylooligosaccharides with low concentration were released from beechwood xylan by reBaxA and DS199 in the presence of reOsXIP. Intrinsic fluorescences of reBaxA and DS199 were statically quenched by rePOsXIP in a concentration-dependent manner. Molecular dynamics (MD) simulations and conformational analysis of OsXIP-BaxA and OsXIP-DS199 revealed that the long loop (Lα4ß5) of OsXIP inserted into the catalytic grooves of BaxA and DS199. The DS199 enhanced the binding affinity to OsXIP, causing conformational alterations on protein-protein interface residues, thereby forming more hydrogen bonds and van der Waals forces. MM/GBSA analysis revealed that the binding free energy (∆Gbind) of OsXIP-DS199 was enhanced by 2.08 kcal/mol compared to that of OsXIP-BaxA. The OsXIP binding induced a conformational changes among residues in the cord and thumb regions of BaxA and DS199. In particular, the T111RYNAP116 residues in the thumb region of DS199 was maintained close to OsXIP by specific bonds. Additional MD simulations revealed that Y113A or T93A mutation of BaxA suppressed the binding affinity by diminishing interface associations of OsXIP-BaxA. This study partially elucidats the molecular basis of inhibitory mechanism and structure-function relationships of GH11 xylanases. Our findings inform rational designs of mutant xylanases with higher resistance to inhibitor proteins.

Experimental and in silico studies of competitive inhibition of family GH10 Aspergillus fumigatus xylanase A by Oryza sativa xylanase inhibitor protein.

The family GH10 Aspergillus fumigatus xylanase A (AfXylA10) gene, afxyla10 was cloned and recombinantly expressed in Pichia pastoris X33. The optimum temperature and pH of reAfXylA10 was 53 °C and 7.0, and Mn2+ remarkably activated the catalytic activity. The recombinant Oryza sativa xylanase inhibitor protein, rePOsXIP significantly inhibited reAfXylA10 with inhibition constant (Ki) of 177.94 nM via competitive inhibition and decreased the concentration of hydrolysate from beechwood xylan. Optimal inhibition of rePOsXIP on reAfXylA10 occurred at 45 °C for 40 min. The fluorescence of reAfXylA10 was statically quenched by rePOsXIP, indicating the formation of reAfXylA10-rePOsXIP complex during their interaction. Furthermore, molecular dynamics (MD) simulations were performed to obtain the detailed information on enzyme-inhibitor interaction. The binding free energy (ΔG) of AfXylA10-OsXIP complex was -30 ± 9 kcal/mol by MM-PBSA calculation, and the α-7 helix of OsXIP anchored in the catalytic cleft of AfXylA10 by competition with the xylan substrate. K239OsXIP stably interacted with the catalytic site E140AfXylA10 through hydrogen bond and vdW interaction. Intermolecular hydrogen bonds T104AfXylA10/V99AfXylA10-Q5OsXIP, R256AfXylA10-E235OsXIP, D155AfXylA10-Y243OsXIP and D145AfXylA10-R194OsXIP on the upper of the TIM barrel were essential for strengthening the stability of complex. Therefore, these non-covalent interactions (NCI) played key role in the interaction between AfXylA10 and OsXIP.