Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Biochar Derived from Post-Adsorbent for Immobilizing Cu and Cd in Sediment: The Effect on Heavy Metal Species and the Microbial Community Composition.

Many biomass wastes or their modified forms have been investigated as heavy metal adsorbents. However, less emphasis has been placed on post-adsorbent management or possible further utilization. In this study, biochar (BC) derived from modified bamboo adsorbent after the adsorption of Cu from an aqueous solution was used for the in situ remediation of lake sediment contaminated with Cd and Cu. The results indicated that the Cu concentration was extremely low (≤0.015 mg/L), while Cd was not detected in the overlying water or the interstitial water after the 90-day BC treatment. The pH value (7.5-8.1) slightly increased, and the toxicity characteristic leaching procedure (TCLP) leachability of the Cu and Cd in the sediment decreased overall. Cu and Cd were preferentially transformed into more stable species. The findings highlighted the potential possibility of BC derived from post-adsorbent being used for sediment remediation. However, the BC addition produced significant effects on the sediment microbial activity and community structure. In general, with an increase in BC, the urease activity increased, while the alkaline phosphatase and invertase activity decreased, which could be attributed to the BC itself. In addition, significant changes in both bacterial and fungal genera were observed. Hence, a cautious approach should be taken in the practical application of BC.

Structural Characterization and In Vitro Antioxidant, Hypoglycemic and Hypolipemic Activities of a Natural Polysaccharide from Liupao Tea.

This study extracted and purified a natural polysaccharide (TPS-5) that has a molecular weight of 48.289 kDa from Liupao tea, a typical dark tea with many benefits to human health. TPS-5 was characterized as a pectin-type acidic polysaccharide. It has a backbone composed of → 2,4)- α- L-Rhap-(1) → 4)- α- D-GalAp-(1) →, with a branch composed of → 5)- α- L-Ara-(1 → 5,3)- α- L-Ara-(1 → 3)- ß- D-Gal-(1 → 3,6)- ß- D-Galp-(1) →. The in vitro biological activity evaluation illustrated that TPS-5 has free radical scavenging, ferric-ion-reducing, digestive enzyme inhibitory, and bile-salt-binding abilities. These results suggest that TPS-5 from Liupao tea has potential applications in functional foods or medicinal products.

The inhibition of macrophage foam cell formation by tetrahydroxystilbene glucoside is driven by suppressing vimentin cytoskeleton.

Macrophage foam cell formation triggered by oxLDL is an important event that occurs during the development of atherosclerosis. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside (TSG) exhibits significant anti-atherosclerotic activity. Herein we used U937 cells induced by PMA and oxLDL in vitro to investigate the inhibitory effects of TSG on U937 differentiation and macrophage foam cell formation. TSG pretreatment markedly inhibited cell differentiation induced by PMA, macrophage apoptosis and foam cell formation induced by oxLDL. The inhibition of vimentin expression and cleavage was involved in these inhibitory effects of TSG. The suppression of vimentin by siRNA in U937 significantly inhibited cell differentiation, apoptosis and foam cell formation. Using inhibitors for TGFßR1 and PI3K, we found that vimentin production in U937 cells is regulated by TGFß/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG pretreatment inhibited both the expression of TGFß1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by PMA and oxLDL. Furthermore, TSG attenuated the induced caspase-3 activation and adhesion molecules levels by PMA and oxLDL. PMA and oxLDL increased the co-localization of vimentin with ICAM-1, which was attenuated by pretreatment with TSG. These results suggest that TSG inhibits macrophage foam cell formation through suppressing vimentin expression and cleavage, adhesion molecules expression and vimentin-ICAM-1 co-localization. The interruption of TGFß/Smad pathway and caspase-3 activation is responsible for the downregulation of TSG on vimentin expression and degradation, respectively.

Tetrahydroxystilbene glucoside inhibits TNF-α-induced migration of vascular smooth muscle cells via suppression of vimentin.

Vascular smooth muscle cell (VSMC) migration triggered by TNF-α is an important event that occurs during the development of atherosclerosis. 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-d-glucoside (TSG) has been proven to exhibit significant anti-atherosclerotic activity. Herein we investigate the inhibitory effect of TSG on TNF-α-induced VSMC migration and explore the underlying mechanisms. TSG pretreatment markedly inhibited TNF-α-induced cell migration. The inhibition of vimentin redistribution and expression was involved in the inhibitory effect of TSG on VSMC migration. The suppression of vimentin expression by shRNA in VSMCs significantly inhibited TNF-α-induced cell migration. Furthermore, TSG inhibited the TNF-α-induced expression of TGFß1 and TGFßR1, and phosphorylation of TGFßR1 and Smad2/3. TSG also suppressed the nuclear translocation of Smad4 induced by TNF-α. These results suggest that TSG inhibits VSMC migration induced by TNF-α through inhibiting vimentin rearrangement and expression. The interruption of TGFß/Smad pathway appears to be responsible for the suppression of TSG on vimentin expression.

Tetrahydroxystilbene glucoside protects against oxidized LDL-induced endothelial dysfunction via regulating vimentin cytoskeleton and its colocalization with ICAM-1 and VCAM-1.

BACKGROUND: Endothelial cell dysfunction triggered by oxidized low-density lipoprotein (oxLDL) is the main event occurring during the development of atherosclerosis. 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (TSG), an active component of the rhizome extract from Polygonum multiflorum, exhibits significant anti-atherosclerotic activity. However, the protective effects of TSG against oxLDL-induced endothelial dysfunction have not been clarified. We investigated the cytoprotective effects of TSG in human umbilical vein endothelial cells (HUVECs) and explored underlying mechanisms. METHODS AND RESULTS: TSG pretreatment markedly attenuated oxLDL-mediated loss of cell viability, release of lactate dehydrogenase (LDH), cell apoptosis, and monocyte adhesion. OxLDL increased vimentin mRNA and protein levels, vimentin cleavage, caspase-3 activation, adhesion molecules levels and their colocalization with vimentin in HUVECs. These alterations were attenuated by pretreatment with TSG. Meanwhile, TSG inhibited both the expression of TGFß1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by oxLDL. Using shRNA, oxLDL-induced cell apoptosis and monocyte adhesion were significantly inhibited by vimentin suppression in HUVECs. CONCLUSIONS: These results suggest that TSG protects HUVECs against oxLDL-induced endothelial dysfunction through inhibiting vimentin expression and cleavage, and the expression of adhesion molecules and their colocalization with vimentin. The interruption of TGFß/Smad pathway and caspase-3 activation appears to be responsible for the downregulation of TSG on vimentin expression and fragmentation, respectively.