A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides.

Carbohydrate-Active enZYme (CAZY) GH89 family enzymes catalyze the cleavage of terminal α-N-acetylglucosamine from glycans and glycoconjugates. Although structurally and mechanistically similar to the human lysosomal α-N-acetylglucosaminidase (hNAGLU) in GH89 which is involved in the degradation of heparan sulfate in the lysosome, the reported bacterial GH89 enzymes characterized so far have no or low activity toward α-N-acetylglucosamine-terminated heparosan oligosaccharides, the preferred substrates of hNAGLU. We cloned and expressed several soluble and active recombinant bacterial GH89 enzymes in Escherichia coli. Among these enzymes, a truncated recombinant α-N-acetylglucosaminidase from gut symbiotic bacterium Bacteroides thetaiotaomicron ∆22Bt3590 was found to catalyze the cleavage of the terminal α1-4-linked N-acetylglucosamine (GlcNAc) from a heparosan disaccharide with high efficiency. Heparosan oligosaccharides with lengths up to decasaccharide were also suitable substrates. This bacterial α-N-acetylglucosaminidase could be a useful catalyst for heparan sulfate analysis.

Recent progress in synthesis of carbohydrates with sugar nucleotide-dependent glycosyltransferases.

Sugar nucleotide-dependent glycosyltransferases (GTs) are key enzymes that catalyze the formation of glycosidic bonds in nature. They have been increasingly applied in the synthesis of complex carbohydrates and glycoconjugates with or without in situ generation of sugar nucleotides. Human GTs are becoming more accessible and new bacterial GTs have been identified and characterized. An increasing number of crystal structures elucidated for GTs from mammalian and bacterial sources facilitate structure-based design of mutants as improved catalysts for synthesis. Automated platforms have also been developed for chemoenzymatic synthesis of carbohydrates. Recent progress in applying sugar nucleotide-dependent GTs in enzymatic and chemoenzymatic synthesis of mammalian glycans and glycoconjugates, bacterial surface glycans, and glycosylated natural products from bacteria and plants are reviewed.

New Means to Control Molecular Assembly.

While self-assembly of molecules is relatively well-known and frequently utilized in chemical synthesis and material science, controlled assembly of molecules represents a new concept and approach. The present work demonstrates the concept of controlled molecular assembly using a non-spherical biomolecule, heparosan tetrasaccharide (MW = 1.099 kD). The key to controlled assembly is the fact that ultra-small solution droplets exhibit different evaporation dynamics from those of larger ones. Using an independently controlled microfluidic probe in an atomic force microscope, sub-femtoliter aqueous droplets containing designed molecules produce well-defined features with dimensions as small as tens of nanometers. The initial shape of the droplet and the concentration of solute within the droplet dictate the final assembly of molecules due to the ultrafast evaporation rate and dynamic spatial confinement of the droplets. The level of control demonstrated in this work brings us closer to programmable synthesis for chemistry and materials science which can be used to develop vehicles for drug delivery three-dimensional nanoprinting in additive manufacturing.

Engineer P. multocida Heparosan Synthase 2 (PmHS2) for Size-Controlled Synthesis of Longer Heparosan Oligosaccharides.

Pasteurella multocida heparosan synthase 2 (PmHS2) is a dual-function polysaccharide synthase having both α1-4-N-acetylglucosaminyltransferase (α1-4-GlcNAcT) and ß1-4-glucuronyltransferase (ß1-4-GlcAT) activities located in two separate catalytic domains. We found that removing PmHS2 N-terminal 80-amino acid residues improved enzyme stability and expression level while retaining its substrate promiscuity. We also identified the reverse glycosylation activities of PmHS2 which complicated its application in size-controlled synthesis of oligosaccharides longer than hexasaccharide. Engineered Δ80PmHS2 single-function-glycosyltransferase mutants Δ80PmHS2_D291N (α1-4-GlcNAcT lacking both forward and reverse ß1-4-GlcAT activities) and Δ80PmHS2_D569N (ß1-4-GlcAT lacking both forward and reverse α1-4-GlcNAcT activities) were designed and showed to minimize side product formation. They were successfully used in a sequential one-pot multienzyme (OPME) platform for size-controlled high-yield production of oligosaccharides up to decasaccharide. The study draws attention to the consideration of reverse glycosylation activities of glycosyltransferases, including polysaccharide synthases, when applying them in the synthesis of oligosaccharides and polysaccharides. The mutagenesis strategy has the potential to be extended to other multifunctional polysaccharide synthases with reverse glycosylation activities to generate catalysts with improved synthetic efficiency.

Application Research on Method of“Role of Identification”in Clinical Teaching / 中国中医药信息杂志

Objective To investigate the score changes of graduates majoring in clinical medicine after using the method of “role of identification”. Methods Sixty students who were undertaking internship in Endocrinology Department of Shanghai Traditional Chinese Medicine Hospital received three-week clinical teaching and examination. Then they got into one-week “role of identification”, and were appointed as clinical teachers to teach what they have learned during the last 3 weeks to the next batch of interns and make another examination. Scores of the two examinations were compared. Results Scores of the examination after received the method in“role of identification”improved significantly (P<0.01). Conclusion The method of“role of identification”can enhance clinical medicine graduates’ mastery of professional knowledge.

Spatial and temporal patterns of primary and secondary syphilis in Shenzhen, China / 中华流行病学杂志

Objective To learn the spatial and temporal patterns of primary syphilis and secondary syphilis in Shenzhen and to provide evidence for carrying out further research on syphilis.Methods Primary syphilis and secondary syphilis cases among residents in Shenzhen between 2005and 2009(n=11 303) were geocoded at street office level (n=55) based on residence at the time of diagnosis. Both spatial and space-time scan statistics were used to identify clusters of street office by using SaTScan software. Results In the purely spatial analyses, clusters were seen in the junction of the Baoan district and Nanshan district (Xinan, Xixiang, Nanshan and Nantou street office) and in the region near Hong Kong (Dongmen, Shekou, and Futian street office), as well as in the other streets where entertainment industry was relatively developed (Longhua, Huafu, Huangbei and Cuizu street office). The clusters had not changed much in the first four years, but nine clusters appeared in 2009.Annually, the most likely clusters were located in Longhua (2005, P≤0.001, RR=3.34), Bamboo (2006, P≤0.001, RR=9.59), Huafu (2007, 2008 years, P≤0.001, RR values were 4.18 and 4.75)and Cuizu (2009, P≤0.001, RR=8.02). In the space-time scan analysis, we found 16 significant clusters, which were similar to the pure spatial analyses. However, regional difference were also found, with the most likely cluster was the Guiyuan street office in 2006. Conclusion Spatial and space-time scan statistics seemed to be effective ways in describing the circular disease clusters. We have had a better understanding on spatial and temporal patterns of primary syphilis and secondary syphilis in Shenzhen through spatial and space-time scan statistics of syphilis surveillance data in the recent years. The changes of spatial and temporal patterns of primary syphilis and secondary syphilis were also described by SaTScan software, which also provided useful reference for the preventive strategies on sexually transmitted diseases as well as on HIV. Useful information was also provided for financial investment and cost-effective studies.

Anticonvulsant valproic acid inhibits cardiomyocyte differentiation of embryonic stem cells by increasing intracellular levels of reactive oxygen species.

BACKGROUND: The anticonvulsant valproic acid (VPA) exerts teratogenic properties and has been demonstrated to cause neural tube defects and malformations of the heart. The effect of VPA on the differentiation of cardiomyocytes from pluripotent murine embryonic stem cells (ES cells) was investigated. METHODS: Embryoid bodies derived from ES cells were treated with different concentrations of VPA and the differentiation of cardiomyocytes was monitored by immunohistochemical staining for sarcomeric alpha-actinin. Cytotoxicity was evaluated by the use of the dead cell stain SYTOX green. Intracellular levels of reactive oxygen species (ROS) within the tissue were evaluated by the use of the redox-sensitive dye dichlorodihydrofluorescein diacetate (H2DCFDA). RESULTS: VPA retarded the growth of ES cell-derived embryoid bodies but did not exert cytotoxic effects. The compound dose-dependently inhibited the development of spontaneously beating clusters of cardiomyocytes within embryoid bodies grown from ES cells and reduced the extension of beating areas of cardiac cells. Furthermore, VPA significantly increased ROS levels, indicating that VPA altered the intracellular redox balance. To investigate whether the inhibition of cardiomyocyte differentiation by VPA was owing to increased ROS overwhelming the intracellular antioxidative defense, the compound was coadministered with the free radical scavenger vitamin E. CONCLUSIONS: This treatment significantly restored cardiomyogenic differentiation, indicating that VPA inhibits cardiomyogenesis of ES cells by increasing intracellular ROS levels.