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Considering the structural complexity of natural bone and the limitations of current treatment options, designing a biomimetic and functional tissue-engineered bone graft has been an urgent need for the replacement and regeneration of defected bone tissue. In light of the cell recruitment to the defect region, scaffold-guided bone tissue engineering has proven to be a viable strategy that is poised to deliver effective osseointegration and vascularization during bone remodeling. Herein, we provide an engineered bone scaffold based on aligned poly(lactic-co-glycolide) (PLGA) nanofibers incorporated with hyaluronic acid oligosaccharide-collagen mineralized microparticles (labeled oHA-Col/HAP) to guide the cell-specific orientation and osseointegration in bone healing. The aligned nanofibers were successfully prepared by a custom-made rotating mandrel with separating railings and HAs-Col/HAP mineralized microparticles were uniformly distributed in the composite scaffolds that acted as temporary templates for bone remodeling. The morphology, physicochemical properties and tensile strength of the scaffolds were characterized, the cell responses and in vivo biocompatibility and biodegradability of the scaffolds were also studied to evaluate the potential for bone tissue engineering. The experimental results illustrated that such anisotropic scaffolds loaded with oHA-Col/HAP microparticles mediated cell orderly arrangement conducive to the migration and recruitment of osseointegration-related cells and were stimulatory of cell proliferation. Those oHA-Col/HAP@PLGA scaffolds exhibited ideal biocompatibility and tissue regenerative capacity in vivo through a higher expression of vascularization-related genes. Overall, the novel engineered bone scaffold promises to serve as alternative candidates for bone tissue engineering applications.

The Report on the Work of the Chinese Government in 2021 emphasised that stable employment is the foundation of national development. Therefore, adjustment of the employment structure is one of the main routes for sustainable development of resource-based cities. However, the impact of employment structure on sustained economic growth, particularly in heterogeneous regions, has not yet been determined. This study analyses China’s employment structure’s spatial evolution, using panel data from 2004 to 2018 of 115 prefecture-level resource-based cities. It explores the driving factors and spatial effects of employment structure changes on economic growth through an extended two-sector economic growth model and spatial econometric model, and proposes solutions for heterogeneous regions. The results show that the labour productivity of the employed population in the secondary industry is the most important factor affecting economic growth, but the spatial effects of employment structure adjustment on economic growth are different in heterogeneous regions. They further reveal that improving the productivity of the employed population in the secondary industry and building an industrial system according to regional advantages are the top priorities for developing the sustainable economy of resource-based cities.

N,N-Diacetylimido protection of 2-aminoglycosides is an elegant strategy but has had limited applications due to unexpected side reactions in glycosylation. We found that high acid concentrations could diminish the side reactions. We observed intermolecular hydrogen bonding among alcohols and acids could disrupt. Assuming that intermolecular hydrogen bonding accelerates the formation of 1,2-orthoamides and disrupting intermolecular hydrogen bonds could turn to the desired glycosylation, we successfully employed sulfenyl triflate pre-activation in the glycosylation of a broad scope of alcohol acceptors, as well as in a one-pot synthesis of a protected human milk oligosaccharide, lacto-N-neotetraose.

Growth driven by innovation is of great importance to help China become a long-term economic force and gain international competitive advantage. Based on evolutionary economic geography theory, this study estimated the related variety and unrelated variety by using four-digit code classified industrial data; it analysed their spatial–temporal evolution and influence on China’s regional innovation levels using prefecture-level city data from 1998 to 2013. Results showed that average levels of related variety increased whereas unrelated variety declined. Regions with high related variety spread from the east coastal region to the central region of China, and regions with high unrelated variety moved from the northeast and north regions to the central region. Related variety promoted innovation by knowledge spillover and produced regional spillover and agglomeration effects among regions with similar levels of economic development. Conversely, unrelated variety hindered the growth of innovation and had no regional spillover among different regions. Technological innovation was the transmission mechanism of growth from related variety to economic development. Thus, it is necessary to strengthen the agglomeration effects of industries with related variety and improve each region’s innovation capacity.

A new two-step enzymatic conversion process for the production of a novel mono-α-1,4-glucosylated rebaudioside A derivative (RA1G) was established via transglycosylation followed by hydrolyzation. In the transglycosylation reaction catalyzed by cyclodextrin glycosyltransferase, rebaudioside A (RA) was converted into glucosylated RA derivatives (RAGs), and the maximum conversion of 87.8% was obtained with the optimal conditions of 2 U/mL enzyme, 82.5 mg/mL β-cyclodextrin, and 82.5 mg/mL RA at 60 °C for 5 h. The obtained RAG solution was then directly used in hydrolyzation. Four amylases were screened for shortening the newly synthesized α-glucans of RAGs. A glucoamylase was found to produce RA1G as the single glucosylated product, and the maximum yield of 53.3% was achieved with the optimal conditions of adding 1.5 U/mL glucoamylase into RAG solution at 60 °C for 3 h. RA1G was identified as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(4-O-α-D-glucopyranosyl-β-D-glucopyranosyl) ester] by MS and NMR analysis and showed an improved sensory quality compared to RA.

β-d-Mannopyranosides are essential components of protein N-glycans, plant β-mannans, and various pathogen surface antigens. Despite their significant biological roles, the formation of 1,2-cis β-ma...

The Chinese economy has developed rapidly since the reform and opening up, but economic growth in Northeast China has declined dramatically after the 21st century. In this context, exploring the characteristics of economic and industrial fluctuations in the northeast of China and their relationship is beneficial to alleviating economic fluctuations and promoting stable economic development from the perspective of industrial development. The relationship between economic and industrial fluctuations in the three provinces of Northeast China was reexamined from the angle of fluctuation components with the complete ensemble empirical mode decomposition with adaptive noise (CEEMDAN) algorithm. The results obtained are as follows: (1) In the three northeastern provinces of China, economic fluctuations were almost free from the influence of the primary industry, most affected by the secondary industry, and gradually influenced by the tertiary industry after the 21st century. (2) Regarding the short-term business cycle of each province, economic development was the most stable when the market and government participated in the development of the secondary industry simultaneously. (3) The midterm business cycle of Jilin Province was affected by the investment of equipment in secondary and tertiary industries, while that of Liaoning Province was affected by the investment of equipment in the secondary industry. (4) Investment in the equipment of the secondary industry and the construction of secondary and tertiary industries was the key to maintaining the stability of long-term business cycle in Heilongjiang Province, and that in the construction of secondary and tertiary industries was the key to maintaining the stability of long-term business cycles in Jilin and Liaoning Provinces.

The current use of synthetic grafts often yields low patency in the reconstruction of small-diameter blood vessels owing to the deposition of thrombi and imperfect coverage of the endothelium on the graft lumen. Therefore, the design of vascular scaffolds with antithrombotic performance and endothelialization is greatly required. Herein, we developed an enzyme-laden scaffold based on hyaluronic acid oligosaccharides-modified collagen nanofibers (labeled HA-COL) to improve the anti-platelet capacity and endothelialization of vascular grafts. In this study, HA-COL nanofibers not only encouraged the endothelialization of vascular scaffolds, but acted as an antiplatelet enzyme-laden platform. Apyrase (Apy) and 5'-nucleotidase (5'-NT) were covalently grafted onto the nanofibers, which in turn converted the platelet-sensitive substance: adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and adenosine, thereby, improving the antithrombotic performance of the scaffolds. Notably, the catalytic end-product: adenosine would work in coordination with HA-COL to synergistically enhance the endothelialization of the vascular scaffolds. The results demonstrated that the enzyme-laden scaffolds maintained catalytic performance, reduced platelet adhesion and aggregation, and guaranteed higher patency after 1-month in situ transplantation. Moreover, these scaffolds showed optimal cytocompatibility, tissue compatibility, scaffold biodegradability and tissue regenerative capability during in vivo implantation. Overall, these engineered vascular scaffolds demonstrated their capacity for endothelialization and antithrombotic performance, suggesting their potential for small-diameter vascular tissue engineering applications. STATEMENT OF SIGNIFICANCE: Considering the critical problems in small-diameter vascular reconstruction, the enzyme-laden vascular scaffolds were prepared for improving in-situ endothelialization and antithrombotic performances of artificial blood vessels. The electrospun HA-COL nanofibers were used as the main matrix materials, which provided favorable structural templates for the regeneration of vasculature and functioned as a platform for the loading of enzymes. The enzyme-laden scaffolds with the biomimetic cascading reaction would convert ADP into adenosine, thereby, decreasing the sensitivity of platelets and improving the antithrombotic performance of tissue-engineered blood vessels (TEBVs). The nanofibrous scaffolds exhibited optimal cytocompatibility, tissue compatibility and regenerative capability, working together with catalytic products of dual-enzyme reaction that would synergistically contribute to TEBVs endothelialization. This study provides a new method for the improvement of in-situ endothelialization of small-diameter TEBVs while qualified with antithrombotic performance.

Stereoselective construction of α-sialyl linkages is one of the most significant challenges in carbohydrate chemistry. In this research, we developed a novel strategy for stereoselective synthesis of α-linked sialosides by protecting the 5-N,4-O-positions of a sialyl donor with an oxazolidinone group and its C-1 carboxylic functionality with a cyanoethyl ester to promote α-glycosylation. We also adopted the more electrophilic N-bromosuccinimide as a promoter to readily activate p-tolyl thiosialoside at -78 °C. The sialylation using this sialyl donor gave excellent yields and α-selectivity. The new synthetic method was used to successfully construct naturally occurring α-sialosides having sialic acid linked to the 6-O- or 3-O-position of galactoside, or 8-O-position of another sialic acid, respectively, as well as other α-linked sialosides.

Northeast China is an important old industrial base in China that is most profoundly affected by the planned economic system. The social and economic development has the characteristics of powerful government and weak market. This study estimates total factor productivity (TFP) utilizing firm-level data of Northeast China, and analyzes the effect of government intervention on TFP growth by applying a spatial econometric model over the period 1998–2007. The results indicate that TFP is gradually increasing and spatially dependents on surrounding areas. Government intervention behavior has a negative effect on TFP in Northeast China. Specifically, the stronger the government’s intervention capacity is, the less TFP is; the higher the environmental connivance is, the less TFP is. Furthermore, government intervention produces adverse spillover effects on surrounding areas, which are caused by imitation and comparison between local governments. We also examine the relationship between traditional factors, including science and technology level, human capital, FDI and openness. The findings reveal that science and technology level, human capital, FDI and openness play positive roles in stimulating TFP growth. Science and technology produce positive spatial spillover on TFP growth, but other factors do not.

Pneumolysin (Ply), pneumococcal surface protein A (PspA), and pneumococcal surface adhesin A (PsaA) are promising cell surface protein antigen targets for Streptococcus pneumoniae (Spn) vaccine development. Herein, we designed and recombined two fusion proteins, named YAPO and YAPL, which contained the main antigenic epitopes of Ply, PspA, and PsaA. In-depth immunological evaluations revealed that YAPO and YAPL had strong immunocompetence to be well-qualified potential carrier proteins. To verify this possibility, a serotype 3 Spn (ST3) CPS pentasaccharide was conjugated to both fusion proteins, respectively, to generate the resultant glycoconjugates. Immunological studies in mice disclosed, as compared with TT conjugate, YAPO and YAPL conjugates provoked robust T-cell dependent immune responses that could provide better recognition, in vitro efficient opsonophagocytosis and in vivo effective protection against various serotypes of Spn. Collectively, YAPO and YAPL were identified as immunopotentiating carriers that could help convert immunologically inactive ST3 pentasaccharide into a T cell-dependent antigen and provide efficient and broad spectrum of immunoprotection coverage so as to formulate functional glycoconjugate vaccines against Spn infections.

Efficient strategies were developed for the synthesis of 6-deoxy-d-manno-heptopyranose and its β-(1 → 3)-linked oligomers as fragments of the common and major capsular polysaccharide, type I O-PS, of Burkholderia pseudomallei and Burkholderia mallei. The unusual heptose was synthesized from mannose, highlighted by the facile Wittig reaction and anti-Markovnikov hydroboration of the resultant olefin. The difficult β-mannosidic linkage in the oligosaccharides was achieved in high stereoselectivity by H-bond-mediated aglycone delivery. All of the oligosaccharides were conjugated with the carrier protein CRM197. Preliminary immunological evaluations of the resultant glycoconjugates in mice verified their efficacy to elicit high titers of immunoglobulin G antibodies and robust T-cell-dependent immune responses. It was also found that the trisaccharide conjugates provoked the strongest immune responses, worthy of further in-depth study for vaccine development.

Glucose-1-phosphate thymidylyltransferase (Cps23FL) from Streptococcus pneumonia serotype 23F is the initial enzyme that catalyses the thymidylyl transfer reaction in prokaryotic deoxythymidine diphosphate-L-rhamnose (dTDP-Rha) biosynthetic pathway. In this study, the broad substrate specificity of Cps23FL towards six glucose-1-phosphates and nine nucleoside triphosphates as substrates was systematically explored, eventually providing access to nineteen sugar nucleotide analogs.

The low-carbon city pilot (LCCP) policy is an important initiative for China to fulfill its international commitment to carbon emission reduction and achieve low-carbon transformation. In this context, this study investigated whether the LCCP policy of China has achieved carbon emission reduction from the production and consumption perspectives and how its underlying mechanism and spatial spillover effect. Using the panel dataset of 285 Chinese prefecture-level cities from 2003 to 2019, this study applied the staggered DID model to examine the effects and its underlying mechanism of the LCCP policy on carbon intensity (CI) and carbon emission per capita (CP). We also conducted heterogeneity and spatial spillover effect analyses using the textual quantification method and spatial DID. Our results show that the LCCP policy effectively reduced CI and CP, but these effects did not appear until the third year of implementation. The above conclusions passed a series of robustness and endogeneity tests. Reducing industrial emissions, improving technological innovation, and optimizing the efficiency of energy usage were three important mechanisms to reduce CI and CP, validating the effectiveness of the LCCP policy. Command-mandatory and voluntary LCCP policy tools achieved better results, and the LCCP policy exerted a significant emission reduction effect on second-tier pilot cities as compared to others. The carbon emission abatement of the LCCP policy has also demonstrated a spatial spillover impact on neighboring cities. This study focused on analyzing the mechanism paths and spatial spillover effects of the LCCP policy impact and provided an important decision-making reference in promoting the LCCP policy for not only China but also other developing countries. Specifically, low-carbon pilot experiences and typical cases should be refined, ways for accelerating the greening and cleaning of energy usage must be explored, and regional joint control and collaborative governance should be established to achieve China's low-carbon transformation.

Group A streptococcus (GAS) is one of the common Gram-positive pathogenic bacteria accounting for a variety of infectious diseases. Currently, there is no commercial vaccine for GAS. To develop efficient GAS vaccines, synthetic tri-, hexa-, and nonasaccharides of a conserved group A carbohydrate (GAC) were conjugated with an inactive mutant of group A streptococcal C5a peptidase (ScpA), ScpA193, to create bivalent conjugate vaccines, which were compared with the corresponding CRM197 and TT conjugates. Systematic evaluations of these semisynthetic conjugates demonstrated that they could induce robust and comparable T-cell-dependent immune responses in mice. It was further disclosed that antibodies provoked by the ScpA193 conjugates, especially that of hexa- and nonasaccharides, could recognize and bind to GAS cells and mediate GAS opsonophagocytosis in vitro. In vivo evaluations of the hexa- and nonasaccharide-ScpA193 conjugates using a mouse model revealed that immunizing mice with especially the latter conjugate could effectively protect the animals from GAS challenges and GAS-induced pulmonary damage and significantly increase animal survival. Further in vitro studies suggested that the two ScpA193 conjugates could function through activating CD4+ T cells and promoting helper T cells (Th) to differentiate into antigen-specific Th1 and Th2 cells. In conclusion, the nonasaccharide-ScpA193 conjugate was identified as a particularly promising GAS vaccine candidate that is worthy of further investigation and development.

Streptococcus pneumoniae serotype 3 (ST3) is one of the main pneumococcal strains that can cause severe invasive diseases, but its current vaccines are relatively inefficient. To develop more effective ST3 vaccines, tetanus toxoid (TT) conjugates of the synthetic penta-, hexa-, hepta-, and octasaccharide analogs of ST3 capsular polysaccharide (CPS) were systematically studied. These conjugates, especially those of penta- and hexasaccharides, were demonstrated to induce extremely robust T cell-dependent immune responses in mouse. Various studies also revealed that the induced antibodies could recognize ST3 CPS and mediate in vitro opsonophagocytic killing of ST3 cells. It was proved ultimately that immunization with the hexasaccharide-TT conjugate could completely protect mice from ST3-caused infection and lung damage and significantly elongate mouse survival. It was proposed that this conjugate functions through the help of CD4+ T cells and via promoting Th cell differentiation into carbohydrate antigen-specific Th2 cells to establish humoral immunity. In conclusion, ST3 CPS hexasaccharide-TT was identified as a particularly promising ST3 vaccine candidate worthy of further investigation and development.

The collagen–hyaluronic acid oligosaccharides (collagen–HA) complex was obtained by covalent attachment using EDC/NHS. The cross-linked product collagen–HA had been confirmed by Fourier transform infrared spectra (FTIR) and the sulfuric acid–carbazole colorimetry, and factors that affect the cross-linking process were investigated. Electrospinning of collagen–HA complex in hexafluoroisopropanol was investigated for the biomimetic nanostructure and biocompatible vascular tissue-engineered inner-layer scaffold. The morphology of the electrospun collagen–HA nanofibers with a series of concentrations was observed by scanning electron microscopy. The thermal behavior and chemical properties of the fabricated scaffolds were characterized using dynamic scanning calorimetry and FTIR after being stabilized by glutaraldehyde vapor. The cell attachment, morphology and proliferation of porcine iliac artery endothelial cells (PIECs) on the electrospun scaffolds were also evaluated. The results indicate that the biomimetic matrix collagen–HA electrospun nanofibrous scaffold could promote PIECs proliferation and may be a better candidate with endothelial potential for the vascular tissue-engineered inner-layer scaffold.

Group A Streptococcus (GAS) cell-wall polysaccharides and streptococcal C5a peptidase (ScpA) are identified as potential target antigens for the development of anti-GAS vaccines. Structurally well-defined mono-, di-, and trimers of the trisaccharide repeating unit of the major and conserved cell-wall polysaccharide of various GAS serotypes were synthesized by a convergent and efficient strategy. These synthetic oligosaccharides, which had a free amino group at the reducing end, were conjugated with a novel ScpA mutant ScpA193 protein through the bifunctional glutaryl linker. The resultant neoglycoproteins were evaluated as conjugate vaccines and compared with other glycoconjugates using the common carrier proteins diphtheria toxin mutant CRM197 and tetanus toxoid (TT). Immunological studies using mice revealed that the ScpA193 conjugates could stimulate not only robust GAS oligosaccharide-specific antibody responses that were comparable to the immunological activities of CRM197 and TT conjugates but also robust ScpA193-specific antibodies, making the ScpA193–oligosaccharide conjugates promising bivalent anti-GAS vaccine candidates.

A new β-rhamnoslytransferase Cps23FT from Streptococcus pneumonia serotype 23F was expressed and characterized. Its enzymatic activity and function were confirmed for the first time by utilizing enzymatically prepared dTDP-Rha and chemically synthesized Glcα-PP-(CH2)11-OPh as substrates. This reaction gave the desired disaccharide Rhaβ-1,4-Glcα-PP-(CH2)11-OPh in a good isolated yield (67%), suggesting the potential of Cps23FT as a tool enzyme for the synthesis of complex oligosaccharides containing difficult β-rhamnosyl linkages. Furthermore, site-directed mutagenesis of Cps23FT disclosed that its 271DKD273 motif was critical for the enzymatic activity and most likely the binding site for the required divalent metal cation.

Group A streptococcal C5a peptidase (ScpA) is a highly conserved surface virulence factor present on group A streptococcus (GAS) cell surfaces. It has attracted much more attention as a promising antigenic target for GAS vaccine development due to its high antigenicity to stimulate specific and immunoprotective antibodies. In this study, a series of segments of ScpA were rationally designed according to the functional domains described in its crystal structure, efficiently prepared and immunologically evaluated so as to assess their potential as antigens for the development of subunit vaccines. Immunological studies revealed that Fn, Fn2, and rsScpA193 proteins were promising antigen candidates worthy for further exploration. In addition, the potential of Fn and Fn2 as carrier proteins to formulate effective glycoconjugate vaccine was also investigated.

In this study, we prepared a biomimetic hyaluronic acid oligosaccharides (oHAs)-based composite scaffold to develop a bone tissue-engineered scaffold for stimulating osteogenesis and endothelialization. The functional oHAs products were firstly synthesized, namely collagen/hyaluronic acid oligosaccharides/hydroxyapatite (Col/oHAs/HAP), chitosan/hyaluronic acid oligosaccharides (CTS/oHAs), and then uniformly distributed in poly (lactic-co-glycolic acid) (PLGA) solution followed by freeze-drying to obtain three-dimensional interconnected scaffolds as temporary templates for bone regeneration. The morphology, physicochemical properties, compressive strength, and degradation behavior of the fabricated scaffolds, as well as in vitro cell responses seeded on these scaffolds and in vivo biocompatibility, were investigated to evaluate the potential for bone tissue engineering. The results indicated that the oHAs-based scaffolds can promote the attachment of endothelial cells, facilitate the osteogenic differentiation of MC3T3-E1 and BMSCs, and have ideal biocompatibility and tissue regenerative capacity, suggesting their potential to serve as alternative candidates for bone tissue engineering applications.

Stereoselective construction of α-sialyl linkages is one of the most significant challenges in carbohydrate chemistry. In this research, we developed a novel strategy for stereoselective synthesis of α-linked sialosides by protecting the 5

Pneumolysin (Ply), pneumococcal surface protein A (PspA), and pneumococcal surface adhesin A (PsaA) are promising cell surface protein antigen targets for

Glucose-1-phosphate thymidylyltransferase (Cps23FL) from

Efficient strategies were developed for the synthesis of 6-deoxy-d

Mycothiol (MSH) is the predominant form of small molecule thiols produced by actinomycetes and plays a pivotal role in the bacterial detoxication process. The only enzyme involved in MSH biosynthes...

Per-O-benzylated ethyl 5-N-acetyl-α-thiosialoside 1 was synthesized and its reactivity as a sialyl donor was investigated. It was demonstrated that the reactions of 1 with various primary and secon...

Trialkylsilane-mediated reductive etherification has been actively used in carbohydrate O-benzylation since it emerged decades ago. In this review, we first trace the origin, development, and possi...

A key target of the overall strategy implementation for regional development since the 18th Party Congress of China has involved taking measures to narrow regional disparities. This is because resource-based cities’ economic development has fallen below general levels due to resource exhaustion and an unbalanced industrial structure, among other factors. Further, an economic gap has long existed between Northeast China’s large number of resource-based cities and non-resource-based cities. This article comprehensively studies the economic convergence of Northeast China’s resource-based cities and non-resource-based cities from 1996 to 2015 by using a dynamic panel to analyze not only the economic development of different industries and types of cities, but also the main factors that influence economic development. The empirical results demonstrate that economic convergence exists in both resource-based and non-resource-based cities, but the economic gap between them has clearly narrowed since the implementation of a strategy to revitalize the Northeast’s old industrial base. Shrinking cities are the fastest to converge, as mature cities are slower and regenerating cities are the slowest; regarding industry structure, the secondary industry dominates the economy in mature and shrinking cities, and the tertiary industry in regenerating cities. The primary stimulus in resource-based cities’ economic development involves upgrading the industrial structure and investing in human capital. As China faces a ‘new normal’ economy, resource-based cities in Northeast China should restructure the economy and perfect their market system to avoid again widening the economic gap.

A monophosphoryl lipid A (MPLA) derivative having the 6′-OH group substituted with an NH2 group was synthesized and coupled with the upstream terminal tetrasaccharide of mycobacterial lipoarabinomannan (LAM) via an amide bond to create a novel type of MPLA-based fully synthetic glycoconjugate vaccine. The same tetrasaccharide was also coupled with MPLA at the 1-O-position. Immunological activities of the two synthetic conjugates were evaluated in mice and compared. Both afforded robust overall and IgG antibody responses, but intraperitoneal injection elicited responses significantly stronger than those from subcutaneous injection. It was thus speculated that MPLA conjugates might act via stimulating B1 lymphocytes present in the intrapleural and peritoneal cavities. Moreover, the 6′-N-conjugate afforded antibody titers much higher than those of the 1-O-conjugate. These results revealed not only the self-adjuvant property of MPLA conjugates to elicit robust IgG antibody responses but also the impact of MPL...

The collagen-chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen-chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen-chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen-chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young's modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen-chitosan nanofiber reinforced by TPU, both the break strength and the Young's modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen-chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.

Two structurally defined, functionalized glycans of lipoteichoic acid (LTA, also known as PS-III) from C. difficile, which have one or two repeating units of LTA linked to the core trisaccharide, were efficiently synthesized via a convergent [2 + 3] or [2 + 2 + 3] strategy. The α-linkage of both N-acetylglucosamine residues in the repeating unit were constructed with glycosyl imidates of azidosugars as donors, while the phosphodiester bridges between the oligosaccharides were fashioned using H-phosphonate chemistry. Both synthetic targets contained a 3-aminopropyl group at the core trisaccharide reducing end, facilitating their conjugation to other biomolecules to afford conjugates useful for various biological studies and applications.

A truncated form (AA32-1032) of group A streptococcus (GAS) C5a peptidase (ScpA), an important GAS virulence factor, and its mutants were prepared and examined to find suitable GAS vaccine candidates and conjugate vaccine carriers. Enzymatic evaluation of the recombinant proteins with a MALDI-TOF MS-based method to analyze the reaction indicated that D130 and N295 were not critical for its activity and S512 was significant but not absolutely required either. Therefore, ΔScpAD130A, ΔScpAN295A and ΔScpAS512A were not suitable vaccine and carrier protein candidates due to their remaining enzymatic activity. A single mutation of H193 to Ala abolished the ScpA activity completely, thereby identifying ΔScpAH193A as a promising candidate that was subjected to immunological studies in mouse. It was shown to elicit high titers of antigen-specific IgG1 antibodies and robust T cell-mediated immunities, verifying its potential as a GAS vaccine. Moreover, conjugating the trisaccharide repeating unit of GAS polysaccharide with ΔScpAH193A could convert the nonimmunogenic oligosaccharide into a highly active and T cell-dependent antigen, demonstrating the potential of ΔScpAH193A as a carrier protein to help formulate robust glycoconjugate vaccines.

Mycothiol (MSH) is the predominant low molecular weight thiol produced by actinomycetes, and it plays a pivotal role in the bacterial detoxication process. 1L-myo-Inositol-1-phosphate (1L-Ins-1-P) α-N-acetylglucosaminyltransferase (GlcNAc-T), known as MshA, is the only glycosyltransferase involved in MSH biosynthesis. In this work, the MshA from Corynebacterium diphtheria, named as CdMshA, was expressed, purified, and studied in detail. Its enzymatic activity to transfer GlcNAc to 1L-Ins-1-P was confirmed by the isolation and rigorous characterization of its reaction product 3-phospho-1-d-myo-inositol-2-acetamido-2-deoxy-α-d-glucopyranoside. CdMshA was shown to accept only UDP-GlcNAc and 1L-Ins-1-P as its substrates among various tested glycosyl donors, such as UDP-GlcNAc, UDP-Gal, UDP-Glc, UDP-GalNAc and UDP-GlcA, and glycosyl acceptors, such as myo-inositol, 1L-Ins-1-P and 1D-Ins-1-P. The results have demonstrated the strict substrate selectivity of CdMshA. Furthermore, its reaction kinetics with UDP-GlcNAc and 1L-Ins-1-P as substrates were characterized, while site-directed mutagenesis of CdMshA disclosed that its amino acid residues N28, K81 and R157 were essential for its enzymatic activity.

Group A

We have recently derived a β-N-acetylhexosaminidase, BbhI, from Bifidobacterium bifidum JCM 1254, which could regioselectively synthesize GlcNAcβ1-3Galβ1-4Glc with a yield of 44.9%. Here, directed evolution of BbhI by domain-targeted mutagenesis was carried out. Firstly, the GH20 domain was selected for random mutagenesis using MEGAWHOP method and a small library of 1300 clones was created. A total of 734 colonies with reduced hydrolytic activity were isolated, and three mutants with elevated transglycosylation yields, GlcNAcβ1-3Galβ1-4Glc yields of 68.5%, 74.7%, and 81.1%, respectively, were obtained. Subsequently, nineteen independent mutants were constructed according to all the mutation sites in these three mutants. After transglycosylation analysis, Asp714 and Trp773 were identified as key residues for improvement in transglycosylation ability and were chosen for the second round of directed evolution by site-saturation mutagenesis. Two most efficient mutants D714T and W773R that acted as trans-β-N-acetylhexosaminidase were finally achieved. D714T with the substitution at the putative nucleophile assistant residue Asp714 by threonine showed high yield of 84.7% with unobserved hydrolysis towards transglycosylation product. W773R with arginine substitution at Trp773 residue locating at the entrance of catalytic cavity led to the yield up to 81.8%. The kcat/Km values of D714T and W773R for hydrolysis of pNP-β-GlcNAc displayed drastic decreases. NMR investigation of protein-substrate interaction revealed an invariable mode of the catalytic cavity of D714T, W773R, and WT BbhI. The collective motions of protein model showed the mutations Thr714 and Arg773 exerted little effect on the dynamics of the inside but a large effect on the dynamics of the outside of catalytic cavity.

α-Amylases are among the most important and widely used industrial enzymes for starch processing. In this work, an α-amylase from Bacillus subtilis XL8 was purified and found to possess both hydrolysis and transglycosylation activities. The optimal pH and temperature for starch hydrolysis were pH 5.0 and 70 °C, respectively. The enzyme could degrade soluble starch into beneficial malto-oligosaccharides ranging from dimer to hexamer. More importantly, it was able to catalyze α-glycosyl transfer from the soluble starch to salidroside, a medicinal plant-derived component with broad pharmacological properties. The transglycosylation reaction catalyzed by the enzyme generated six derivatives in a total high yield of 73.4% when incubating with 100 mg/mL soluble starch and 50 mM salidroside (pH 7.5) at 50 °C for 2 h. These derivatives were identified as α-1,4-glucosyl, maltosyl, maltotriosyl, maltotetraosyl, maltopentaosyl, and maltohexaosyl salidrosides, respectively. They were novel promising compounds that might integrate the bioactive functions of malto-oligosaccharides and salidroside.