Mechanism of secretion of TcpF by the Vibrio cholerae toxin-coregulated pilus.

Many bacteria possess dynamic filaments called Type IV pili (T4P) that perform diverse functions in colonization and dissemination, including host cell adhesion, DNA uptake, and secretion of protein substrates-exoproteins-from the periplasm to the extracellular space. The Vibrio cholerae toxin-coregulated pilus (TCP) and the enterotoxigenic Escherichia coli CFA/III pilus each mediates export of a single exoprotein, TcpF and CofJ, respectively. Here, we show that the disordered N-terminal segment of mature TcpF is the export signal (ES) recognized by TCP. Deletion of the ES disrupts secretion and causes TcpF to accumulate in the V. cholerae periplasm. The ES alone can mediate export of Neisseria gonorrhoeae FbpA by V. cholerae in a T4P-dependent manner. The ES is specific for its autologous T4P machinery as CofJ bearing the TcpF ES is exported by V. cholerae, whereas TcpF bearing the CofJ ES is not. Specificity is mediated by binding of the ES to TcpB, a minor pilin that primes pilus assembly and forms a trimer at the pilus tip. Finally, the ES is proteolyzed from the mature TcpF protein upon secretion. Together, these results provide a mechanism for delivery of TcpF across the outer membrane and release into the extracellular space.

Myricetin Nanofibers Enhanced Water Solubility and Skin Penetration for Increasing Antioxidant and Photoprotective Activities.

Excessive exposure to ultraviolet radiation (UV) can induce oxidative stress through the over-production of reactive oxygen species (ROS) on the skin. Myricetin (MYR), a natural flavonoid compound, significantly inhibited UV-induced keratinocyte damage; however, its bioavailability is limited by its poor water solubility and inefficient skin penetration ability, which subsequently influences its biological activity. The purpose of the study was to develop a myricetin nanofibers (MyNF) system of hydroxypropyl-ß-cyclodextrin (HPBCD)/polyvinylpyrrolidone K120 (PVP)-loaded with MYR that would enhance the water solubility and skin penetration by changing the physicochemical characteristics of MYR, including reducing the particle size, increasing the specific surface area, and amorphous transformation. The results also revealed that the MyNF can reduce cytotoxicity in HaCaT keratinocytes when compared with MYR; additionally, MyNF had better antioxidant and photoprotective activity than raw MYR for the UVB-induced HaCaT keratinocytes damage model due to the MyNF increased water solubility and permeability. In conclusion, our results demonstrate that MyNF is a safe, photostable, and thermostable topical ingredient of antioxidant nanofibers to enhance the skin penetration of MYR and prevent UVB-induced skin damage.

Liposomes Encapsulating Morin: Investigation of Physicochemical Properties, Dermal Absorption Improvement and Anti-Aging Activity in PM-Induced Keratinocytes.

Recently, a global market for anti-aging skin care using botanicals has been noticeably developing. Morin, 3,5,7,2',4'-pentahydroxyflavone, is a polyphenol with many pharmacological properties including antioxidant, anti-inflammation and photoprotection. However, poor aqueous solubility of morin restricts its application in pharmaceuticals. The present study aimed to encapsulate morin into liposomal vesicles to improve its water solubility and skin penetration, and further investigated its ROS inhibition and anti-aging activity in HaCaT keratinocytes induced by particulate matters (PMs). Our data presented that morin was a strong DPPH• radical scavenger. Morin displayed a remarkable ROS inhibitory ability and protected keratinocytes against PMs by downregulating matrix metalloproteinase-1 (MMP-1) expression via suppressing p-ERK and p-p38 in the MAPK pathway. Moreover, water solubility of liposomal morin (LM) prepared by the thin film hydration method was significantly better than free form of morin due to particle size reduction of LM. Our results also demonstrated that deformable liposomal vesicles were achieved for increasing dermal absorption. Additionally, LM (morin:lecinolws-50:tween-80:PF-68, 1:2.5:2.5:5) was able to effectively reduce generation of ROS, inactivate p-ERK, p-p38 and MMP-1 in HaCaT cells exposed to PM. In conclusion, our findings suggested that LM would be a bright candidate for various topical anti-aging and anti-pollution products.

DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform.

Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 µL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 µL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept.

Extraction of Cell-free Dna from An Embryo-culture Medium Using Micro-scale Bio-reagents on Ewod.

As scientific and technical knowledge advances, research on biomedical micro-electromechanical systems (bio-MEMS) is also developing towards lab-on-a-chip (LOC) devices. A digital microfluidic (DMF) system specialized for an electrowetting- on-dielectric (EWOD) mechanism is a promising technique for such point-of-care systems. EWOD microfluidic biochemical analytical systems provide applications over a broad range in the lab-on-a-chip field. In this report, we treated extraction of cell-free DNA (cf-DNA) at a small concentration from a mouse embryo culture medium (2.5 days & 3.5 days) with electro-wetting on a dielectric (EWOD) platform using bio-reagents of micro-scale quantity. For such extraction, we modified a conventional method of genomic-DNA (g-DNA) extraction using magnetic beads (MB). To prove that extraction of cf-DNA with EWOD was accomplished, as trials we extracted designed-DNA (obtained from Chang Gung Memorial Hospital (CGMH), Taiwan which shows properties similar to that of cf-DNA). Using that designed DNA, extraction with both conventional and EWOD methods has been performed; the mean percentage of extraction with both methods was calculated for a comparison. From the cycle threshold (Ct) results with a quantitative polymerase chain reaction (q-PCR), the mean extraction percentages were obtained as 14.8 percent according to the conventional method and 23 percent with EWOD. These results show that DNA extraction with EWOD appears promising. The EWOD extraction involved voltage 100 V and frequency 2 kHz. From this analysis, we generated a protocol for an improved extraction percentage on a EWOD chip and performed cf-DNA extraction from an embryo-culture medium (KSOM medium) at 3.5 and 2.5 days. The mean weight obtained for EWOD-extracted cf-DNA is 0.33 fg from the 3.5-day sample and 31.95 fg from the 2.5-day sample. All these results will pave a new path towards a renowned lab-on-a-chip concept.

Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice.

Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the "PICKY" software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients.

Biochemical characterization and structural analysis of a bifunctional cellulase/xylanase from Clostridium thermocellum.

We expressed an active form of CtCel5E (a bifunctional cellulase/xylanase from Clostridium thermocellum), performed biochemical characterization, and determined its apo- and ligand-bound crystal structures. From the structures, Asn-93, His-168, His-169, Asn-208, Trp-347, and Asn-349 were shown to provide hydrogen-bonding/hydrophobic interactions with both ligands. Compared with the structures of TmCel5A, a bifunctional cellulase/mannanase homolog from Thermotoga maritima, a flexible loop region in CtCel5E is the key for discriminating substrates. Moreover, site-directed mutagenesis data confirmed that His-168 is essential for xylanase activity, and His-169 is more important for xylanase activity, whereas Asn-93, Asn-208, Tyr-270, Trp-347, and Asn-349 are critical for both activities. In contrast, F267A improves enzyme activities.

Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.

Improving the specific activity of ß-mannanase from Aspergillus niger BK01 by structure-based rational design.

ß-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic ß-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (ß/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.

Structural and mutagenetic analyses of a 1,3-1,4-ß-glucanase from Paecilomyces thermophila.

The thermostable 1,3-1,4-ß-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley ß-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80Å to 2.25Å resolution. PtLic16A adopts a typical ß-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3-1,4-ß-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3-1,4-ß-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.

Studies of efficacy and liver toxicity related to adeno-associated virus-mediated RNA interference.

Adeno-associated virus (AAV)-mediated RNA interference shows promise as a therapy for chronic hepatitis B virus (HBV) infection, but its low efficacy and hepatotoxicity pose major challenges. We have generated AAV vectors containing different promoters and a panel of HBV-specific short hairpin RNAs (shRNAs) to investigate factors that contribute to the efficacy and pathogenesis of AAV-mediated RNA interference. HBV transgenic mice injected with high doses of AAV vectors containing the U6 promoter produced abundant shRNAs, transiently inhibited HBV, but induced severe hepatotoxicity. Sustained HBV suppression without liver toxicity can be achieved by lowering the dose of AAV-U6 vectors. AAVs containing the weaker H1 promoter did not cause liver injury, but their therapeutic efficacy was highly dependent on the sequence of the shRNA. Mice treated with the toxic U6-promoter-driven shRNA showed little change in hepatic microRNA levels, but a dramatic increase in hepatic leukocytes and inflammatory cytokines and chemokines. Hepatotoxicity was completely absent in immunodeficient mice and significantly alleviated in wild-type mice depleted of macrophages and granulocytes, suggesting that host inflammatory responses are the major cause of liver injury induced by the overexpressed shRNAs from AAV-U6 vectors. Our results demonstrate that selection of a highly potent shRNA and control its expression level is critical to achieve sustained HBV suppression without inducing inflammatory side effects.

Characteristics and antioxidant activities of silymarin nanoparticles.

Silymarin, a well known hepatoprotective drug, has been routinely used in treating liver disorders. However, its bioavailability and therapeutic efficiency are limited by the poor aqueous solubility. In this study, we used the nanoprecipitation technique to develop a nanoparticles system to improve the solubility of silymarin. The newly developed silymarin nanoparticles were characterized for mean particle size, morphology, intermolecular interaction, crystalline features and dissolution property, as well as assessing for antioxidant activities. Results indicated that a drastic change in the physiochemical properties of silymarin was noted in the form of nanoparticles, as displayed by the extremely small particle size (46.1 +/- 1.73 nm), the formation of intermolecular hydrogen bonding between silymarin and matrix of nanoparticles, and the rendering of amorphous state. These phenomena have contributed to the enhanced dissolution property of silymarin nanoparticles, as well as a greater potency in DPPH radical scavenging, anti-superoxide anion formation, and superoxide anion scavenging activities than the crude silymarin. The present study concludes that silymarin nanoparticles have an improved physicochemical property as demonstrated by an increased solubility and enhanced antioxidant activities.

Green tea catechins decrease oxidative stress in surgical menopause-induced overactive bladder in a rat model.

UNLABELLED: What's known on the subject? and What does the study add? Ovary hormone deficiency and the age-related changes in post-menopausal women are subjected to a number of urological dysfunctions, including overactive bladder syndrome. Green tea is a popular healthy drink worldwide and its extract catechin has strong anti-inflammatory and antioxidant properties. EGCG, the major type of catechin, is an antioxidant polyphenol flavonoid isolated from green tea. EGCG supplement could prevent ovariectomy-induced bladder dysfunction in a dose-related manner through its anti-oxidant, anti-fibrosis and anti-apoptosis effects. OBJECTIVE: To evaluate whether green tea extract, epigallocatechin gallate (EGCG), could prevent ovariectomy-induced overactive bladder (OAB) and to investigate its antioxidant, anti-apoptotic and anti-fibrosis effects. MATERIALS AND METHODS: In all, 48 female Sprague-Dawley rats were divided into four groups. After bilateral ovariectomy, the first group served as the ovariectomy control, the second group received EGCG 1 µM/kg daily i.p. injection after ovariectomy surgery, and the third group received EGCG 10 µM/kg daily i.p. injection. The fourth group was taken as the sham without ovariectomy surgery. The rats were killed after 6 months after ovariectomy surgery. Cystometrograms were performed for the measure of bladder overactivity. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay was used to evaluate apoptotic cells. Western immunoblots were performed to determine the expressions of inflammatory markers, apoptosis-associated proteins and oxidative stress markers. RESULTS: Long-term ovariectomy significantly increased non-voiding contractions and decreased bladder compliance. Treatment with EGCG significantly increased bladder compliance and diminished non-voiding contractions. Ovariectomy significantly increased apoptotic cells and enhanced interstitial fibrosis in bladders. The expression of caspase-3 significantly increased, while that of Bcl-2 notably decreased after ovariectomy. Inflammatory and fibrosis markers, TGF-ß, fibronectin and type I collagen expressions were significantly increased after 6 months of ovariectomy surgery. Treatment with EGCG significantly decreased TGF-ß and type I collagen expressions. Oxidative stress markers, nitrotyrosine and protein carbonylation levels were significantly increased in the ovariectomy group. EGCG could attenuate this oxidative damage in dose-dependent fashion. CONCLUSIONS: Ovariectomy increased oxidative damage, enhanced voiding frequency and decreased bladder compliance. EGCG could restore ovariectomy-induced bladder dysfunction in a dose-dependent fashion through antioxidant, anti-fibrosis and anti-apoptosis effects.

Resveratrol nanoparticle system improves dissolution properties and enhances the hepatoprotective effect of resveratrol through antioxidant and anti-inflammatory pathways.

Resveratrol (RES), a well-known antioxidant and anti-inflammatory compound, is abundant in red wine and exerts numerous pharmacological effects, including hepatoprotection and cadioprotection. Unfortunately, RES is restricted in clinical application due to poor dissolution property and adsorption. In addition, red wine as a supplement for preventing disease is not recommended for patients with alcohol-related disorders. To address these limitations, we successfully developed a novel RES nanoparticle system (RESN) and demonstrated that RESN could circumvent the physicochemical drawbacks of raw RES with respect to dissolution, such as the reduction of particle size, amorphous transformation, and hydrogen-bond formation. In addition, we employed an animal model of CCl4-induced hepatotoxicity to estimate the potential of the nanoparticle formulation to improve the hepatoprotective effect of orally administered RES. Our results demonstrated that RESN can diminish liver function markers (aspartate aminotransferase and alanine aminotransferase) by decreasing hepatocyte death due to CCl4-induced hepatotoxicity in rats, when compared with RES administration. The effect was achieved by reducing oxidative stress (decreased reactive oxygen species and lipid peroxidation) and lowering inflammatory cytokines (decreased tumor necrosis factor-α and interleukin 1ß) and protein expression (cyclooxygenase-2, inducible nitric oxide synthase, cytosolic phospholipase A2, and caspase-3). In conclusion, enhancement of the dissolution of RES through a nanoparticle engineering process can result in increased hepatoprotective effects mediated by antioxidant and anti-inflammatory activities. Consequently, we suggest that RESN deserves further study, perhaps in prophylaxis of chronic liver diseases.

Enhanced activity of Thermotoga maritima cellulase 12A by mutating a unique surface loop.

Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable ß-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using site-directed mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The k (cat) and K (m) values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application.

Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose.

The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29Å to 2.40Å resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers ß-glucosyl and ß-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues.

Crystal structures of Bacillus alkaline phytase in complex with divalent metal ions and inositol hexasulfate.

Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.

Enhancement of dissolution and antioxidant activity of kaempferol using a nanoparticle engineering process.

Kaempferol (KAE) is a strong antioxidant flavonoid compound, but its clinical application is limited by quantity and poor dissolution property. However, the dissolution mechanism of a kaempferol nanoparticle formulation (KAEN) has not yet been elucidated. The aim of the present study was therefore to use a nanoparticle engineering process to resolve the dissolution problem. Our data indicated that KAEN effectively increased the dissolution percentage by particle size reduction, high encapsulation efficiency, amorphous transformation, and hydrogen-bond formation with excipients. In addition, we used several different antioxidant activity assays to evaluate KAE and KAEN. The data indicated that KAEN retained potent antioxidant activity after the nanoparticle engineering process and showed better antioxidant activity than KAE dissolved in water (P < 0.05). According to these findings, we concluded that KAEN could be a low-dose alternative to KAE in health food and future clinical research.

Crystal structure and substrate-binding mode of cellulase 12A from Thermotoga maritima.

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a ß-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a ß-jellyroll protein fold typical of the GH12-family enzymes, with two curved ß-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted ß-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure.

Curcumin nanoparticles improve the physicochemical properties of curcumin and effectively enhance its antioxidant and antihepatoma activities.

Curcumin (CUR), a natural polyphenol isolated from tumeric ( Curcuma longa ), has been documented to possess antioxidant and anticancer activities. Unfortunately, the compound has poor aqueous solubility, which results in poor bioavailability following high doses by oral administration. To improve the solubility of CUR, we developed a novel curcumin nanoparticle system (CURN) and investigated its physicochemical properties as well as its enhanced dissolution mechanism. Our results indicated that CURN improved the physicochemical properties of CUR, including a reduction in particle size and the formation of an amorphous state with hydrogen bonding, both of which increased the drug release of the compound. Moreover, in vitro studies indicated that CURN significantly enhanced the antioxidant and antihepatoma activities of CUR (P < 0.05). Consequently, we suggest that CURN can be used to reduce the dosage of CUR and improve its bioavailability and merits further investigation for therapeutic applications.