Enzymatic construction of a library of even- and odd-numbered heparosan oligosaccharides and their N-sulfonated derivatives.

Low-molecular-weight heparins (LMWHs), especially the specific-sized heparin oligosaccharides, are attractive for the therapeutic applications, while their synthesis remains challenging. In the present study, unsaturated even-numbered heparosan oligosaccharides were firstly prepared by cleaving high-molecular-weight heparosan using recombinant heparinase III (HepIII). The conversion rates of the unsaturated disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides were 33.9 %, 47.9 %, 78.7 %, 71.8 %, and 53.4 %, respectively. After processing the aforementioned heparosan oligosaccharides with the Δ4,5 unsaturated glycuronidase, saturated odd-numbered heparosan trisaccharides, pentasaccharides, heptasaccharides, and nonasaccharides were produced. It was observed that among them, the pentasaccharides were the smallest units of saturated odd-numbered oligosaccharides recognized by HepIII. These oligosaccharides were further catalyzed with bifunctional heparan sulfate N-deacetylase/N-sulfotransferase (NDST) under optimized reaction conditions. It was found that the tetrasaccharide was defined as the smallest recognition unit for NDST, obtaining the N-sulfonated heparosan tetrasaccharides, pentasaccharides, and hexasaccharides with a single sulfonate group, as well as N-sulfonated heparosan heptasaccharides, octasaccharides, and nonasaccharides with multiple sulfonate groups. These results provide an easy pathway for constructing a library of specific-sized N-sulfonated heparosan oligosaccharides that can be used as the substrates for the enzymatic synthesis of LMWHs and heparin oligosaccharides, shedding new light on the substrate preference of NDST.

Production of different molecular weight glycosaminoglycans with microbial cell factories.

Glycosaminoglycans (GAGs) are naturally occurring acidic polysaccharides with wide applications in pharmaceuticals, cosmetics, and health foods. The diverse biological activities and physiological functions of GAGs are closely associated with their molecular weights and sulfation patterns. Except for the non-sulfated hyaluronan which can be synthesized naturally by group A Streptococcus, all the other GAGs such as heparin and chondroitin sulfate are mainly acquired from animal tissues. Microbial cell factories provide a more effective platform for the production of structurally homogeneous GAGs. Enhancing the production efficiency of polysaccharides, accurately regulating the GAGs molecular weight, and effectively controlling the sulfation degree of GAGs represent the major challenges of developing GAGs microbial cell factories. Several enzymatic, metabolic engineering, and synthetic biology strategies have been developed to tackle these obstacles and push forward the industrialization of biotechnologically produced GAGs. This review summarizes the recent advances in the construction of GAGs synthesis cell factories, regulation of GAG molecular weight, and modification of GAGs chains. Furthermore, the challenges and prospects for future research in this field are also discussed.

Improvement of the stability and catalytic efficiency of heparan sulfate N-sulfotransferase for preparing N-sulfated heparosan.

The chemo-enzymatic and enzymatic synthesis of heparan sulfate and heparin are considered as an attractive alternative to the extraction of heparin from animal tissues. Sulfation of the hydroxyl group at position 2 of the deacetylated glucosamine is a prerequisite for subsequent enzymatic modifications. In this study, multiple strategies, including truncation mutagenesis based on B-factor values, site-directed mutagenesis guided by multiple sequence alignment, and structural analysis were performed to improve the stability and activity of human N-sulfotransferase. Eventually, a combined variant Mut02 (MBP-hNST-NΔ599-602/S637P/S741P/E839P/L842P/K779N/R782V) was successfully constructed, whose half-life at 37°C and catalytic activity were increased by 105-fold and 1.35-fold, respectively. After efficient overexpression using the Escherichia coli expression system, the variant Mut02 was applied to N-sulfation of the chemically deacetylated heparosan. The N-sulfation content reached around 82.87% which was nearly 1.88-fold higher than that of the wild-type. The variant Mut02 with high stability and catalytic efficiency has great potential for heparin biomanufacturing.

Construction of Osmotic Pressure Responsive Vacuole-like Bacterial Organelles with Capsular Polysaccharides as Building Blocks.

Many artificial organelles or subcellular compartments have been developed to tune gene expression, regulate metabolic pathways, or endow new cell functions. Most of these organelles or compartments were built using proteins or nucleic acids as building blocks. In this study, we demonstrated that capsular polysaccharide (CPS) retained inside bacteria cytosol assembled into mechanically stable CPS compartments. The CPS compartments were able to accommodate and release protein molecules but not lipids or nucleic acids. Intriguingly, we found that the CPS compartment size responds to osmotic stress and this compartment improves cell survival under high osmotic pressures, which was similar to the vacuole functionalities. By fine-tuning the synthesis and degradation of CPS with osmotic stress-responsive promoters, we achieved dynamic regulation of the size of CPS compartments and the host cells in response to external osmotic stress. Our results shed new light on developing prokaryotic artificial organelles with carbohydrate macromolecules.

SVM based imbalanced correction method for Power Systems Transient stability evaluation.

Due to the requirement of safety and reliability in power systems, unstable samples in the real system are rarely appeared. The evaluation results of the model trained by these imbalance samples have a certain preference. Generally, the imbalance in quantity is taken into account, while the imbalance in quality is ignored. Faced with such a problem, an imbalanced correction method based on support vector machine (SVM) is proposed. Firstly, the classification hyperplane trained by SVM and the normalized Euclidean distance between each sample and the classification hyperplane are calculated so as to obtain their fault severity. Based on this, training samples can be grouped to multilevel sets. Then, the original stacked sparse auto-encoder (SSAE) are pretrained to quantify the imbalance between two classes of samples in multilevel sets. Subsequently, in order to improve the imbalance of training samples, a cost-sensitive correction matrix is generated according to the imbalanced information of multilevel sets. Finally, the loss function of SSAE is modified by cost-sensitive correction matrix to establish the final classifier. Simulation results in IEEE 39-bus system and the realistic regional power system of Eastern China show the high performance of the proposed imbalanced correction method.

Biosynthesis of non-sulfated high-molecular-weight glycosaminoglycans and specific-sized oligosaccharides.

Non-sulfated forms of glycosaminoglycans (NSGAGs) including hyaluronan, chondroitin and heparosan with high-molecular-weight (HMW) are extensively used biomaterials, while NSGAGs oligosaccharides display strong bioactivities. However, microbial production of HMW-NSGAGs and oligosaccharides with specific size are always challenging. Here, a membrane shield strategy was developed to produce HMW-NSGAGs by recruiting type II NSGAG synthases in Corynebacterium glutamicum. By enhancing precursor supplies and reinforcing cell membrane, the MWs of hyaluronan, heparosan and chondroitin reached 4100 kDa, 3000 kDa and 2400 kDa, respectively. In parallel, a synchronized depolymerization-polymerization strategy was developed by co-expressing NSGAGs synthases and lyases. With cell membrane as a filter, we achieved the direct biosynthesis of NSGAGs tetrasaccharides and disaccharides. The titers of chondroitin, hyaluronan and heparosan tetrasaccharides and disaccharides reached 10.9 g L-1, 12.1 g L-1 and 5.8 g L-1, respectively. The strategies developed here should also be applicable to the biosynthesis of other polysaccharides.

Enzymatic Production of Chondroitin Oligosaccharides and Its Sulfate Derivatives.

Chondroitin sulfate (CS) has a wide range of physiological functions and clinical applications. However, the biosynthesis of chondroitin oligosaccharides (o-CHs) and sulfate derivatives with specific length is always challenging. Herein, we report enzymatic strategies for producing homogeneous o-CHs and its sulfate derivatives from microbial sourced chondroitin. Chondroitin disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides with defined structure were produced by controllably depolymerizing microbial sourced chondroitin with an engineered chondroitinase ABC I. The highest conversion rates of the above corresponding o-CHs were 65.5%, 32.1%, 12.7%, 7.2%, and 16.3%, respectively. A new efficient enzymatic sulfation system that directly initiates from adenosine 5'-triphosphate (ATP) and sulfate was developed and improved the sulfation of chondroitin from 8.3% to 85.8% by optimizing the temperature, sulfate and ATP concentration. o-CHs decasaccharide, octasaccharide, hexasaccharide, tetrasaccharide and disaccharide were modified and the corresponding sulfate derivatives with one sulfate group were prepared. The enzymatic approaches constructed here for preparing o-CHs and its sulfate derivatives pave the way for the study of structure-activity relationship and applications.

Engineering the probiotic bacterium Escherichia coli Nissle 1917 as an efficient cell factory for heparosan biosynthesis.

Heparosan as an acidic polysaccharide is mainly applied for heparin biosynthesis and drug delivery. Escherichia coli Nissle 1917 (EcN) naturally synthesizes and secrets heparosan as its capsular polysaccharides. In this study, we described the metabolic engineering of EcN to enhance heparosan production by optimizing the biosynthesis of precursors UDP-GlcA and UDP-GlcNAc and the expression of heparosan synthase. The orthologs of heparosan synthetic pathway enzymes from five species were expressed and comparatively investigated. bsGalU and ecKfiD for UDP-GlcA and ecGlmM for UDP-GlcNAc were introduced into EcN and the production of heparosan was increased from 0.15 g/L to 0.34 g/L, 0.39 g/L and 0.37 g/L, respectively. Combinational overexpression of bsGalU, ecKfiD and ecGlmM improved heparosan production to 0.80 g/L in flask cultures. After further upregulation of the endogenous heparosan synthases KfiAC, the titer of heparosan was improved to 1.29 g/L. Meanwhile, pathway engineering also led to the fluctuation of molecular weights between 312.39 and 410.84 kDa. Eventually, the engineered strain EC048 with overexpression of bsGalU, ecKfiD, ecGlmM and KfiAC produced 11.50 g/L heparosan in 3-L fed-batch fermentor, demonstrating EcN as a good microbial chassis is applicable for engineering an efficient heparosan cell factory.

Eliminating the capsule-like layer to promote glucose uptake for hyaluronan production by engineered Corynebacterium glutamicum.

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L-1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum, changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L-1. Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers.

Engineering strong and stress-responsive promoters in Bacillus subtilis by interlocking sigma factor binding motifs.

Prokaryotic gene expression is largely regulated on transcriptional levels with the involvement of promoters, RNA polymerase and sigma factors. Developing new promoters to customize gene transcriptional regulation becomes increasingly demanded in synthetic biology and biotechnology. In this study, we designed synthetic promoters in the Gram-positive model bacterium Bacillus subtilis by interlocking the binding motifs of σA for house-keeping gene expression and that of two alternative sigma factors σH and σB which are involved in responding post-exponential growth and general stress, respectively. The developed promoters are recognized by multiple sigma factors and hence generate strong transcriptional strength when host cells grow under normal or stressed conditions. With green fluorescent protein as the reporter, a set of strong promoters were identified, in which the transcription activities of PHA-1, PHAB-4, PHAB-7 were 18.6, 4.1, 3.3 fold of that of the commonly used promoter P43, respectively. Moreover, some of the promoters such as PHA-1, PHAB-4, PHAB-7, PBA-2 displayed increased transcriptional activities in response to high salinity or low pH. The promoters developed in this study should enrich the biotechnological toolboxes of B. subtilis.

Increased levels of serum histone H4 and activated protein C in patients with active rheumatoid arthritis.

OBJECTIVES: We aimed to examine the levels of serum H4 and activated protein C (APC) in rheumatoid arthritis (RA) and other autoimmune conditions, and investigate the associations between H4 or APC levels and disease activity indicators in RA. METHODS: Serum H4 and APC distribution was examined in samples from patients with RA, systemic lupus erythematosus (SLE), polymyositis (PM), and ankylosing spondylitis (AS), as well as in samples from healthy controls, using commercial ELISA kits. Associations of serum H4 or APC levels with disease variables in patients with RA were evaluated. Receiver operating characteristic (ROC) curve analysis was performed to assess the discriminant capacity of APC against RA and non-RA. RESULTS: The patients with RA, PM, and AS showed higher serum levels of H4 and APC than those from the healthy control individuals, while the SLE patients showed higher serum levels of APC only. Moderate positive correlations between H4 levels and absolute neutrophil count (ANC), platelet count (PLT), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), brinogen (FIB), D-dimer (DD), and complement fraction 3 (C3) were observed. Positive correlations between APC levels and PLT, RF, DD, or DAS28 were additionally found. ROC curve analysis revealed that APC discriminated well between RA and non-RA. CONCLUSIONS: H4 and APC concentrations are elevated in patients with chronic in ammatory autoimmune diseases. The observed associations between H4 and APC and disease variables in patients with RA support a role for H4 and APC in the in ammatory process of the disease.

Maskless fabrication of slanted annular aperture arrays.

We report maskless fabrication of high-aspect-ratio slanted annular aperture arrays (SAAAs) in gold films using focused ion beam lithography. By tilting the substrate, SAAAs with the desired tilting angle can be fabricated. Our experimental results demonstrate accurate control over aperture size, obliqueness, and reproducibility. We also show that the resulted plasmonic resonances of SAAAs can be effectively tuned via obliqueness control. This versatile approach may enable fabrication of more complicated plasmonic nanostructures. The demonstrated gold SAAAs could also find many potential applications in plasmon-assisted sensing and surface enhanced spectroscopy.

Design of Analytical Run Length for Clinical Chemistry Analytes.

BACKGROUND: The expected number of unacceptable patient results E (Nu) can be set as a patient-based quality goal. Analytical run length can be designed to limit E (Nu) < 1. METHODS: The new internal quality control (IQC) strategy and length of analytical run for each analyte was applied to routine IQC paralleled with the way before redesign. IQC charts were produced by QC test results to analyze and compare the performance of out-of-control error detection. RESULTS: Optimal analytical run lengths designed by the quality control computer software QCCS 2008 were 39 for albumin, 61 for cholesterol, 900 for triglycerides, 112 for aspartate aminotransferase, 279 for lactate dehydrogenase, 267 for alcaline phospatase, 363 for total bilirubin, 151 for ceatinine, 230 for uric acid, 46 for phosphorus PHOS, 158 for carbon dioxide, and 580 for glucose. After being redesigned, IQC strategies for ALB, CHOL, and PHOS detected more out-of-control error than before and achieved more cost-effectiveness. CONCLUSIONS: Using E (Nu) as a QC performance measure, frequency of QC testing can be objectively designed. Additionally, new QC strategies can help find more problems of testing systems and promote efficiency and cost savings.

Internal quality control practice of thyroid disease related tests and imprecision analysis in China.

BACKGROUND: Internal quality control (IQC) has a long and well-established role in clinical laboratories and the quality of laboratories has achieved great improvement in China. However, the practice of IQC varies significantly between institutions and many problems still exists. Consequently, the Chinese National Center for Clinical Laboratories has been undertaking monthly nation-wide surveys of current IQC practice of tests since 2010 to monitor laboratory quality. Thyroid disease related tests were chosen for this research. METHODS: Different numbers of laboratories in China participating national external quality assessment (EQA) schemes of Total Thyroxine (TT4), Total Triiodothyronine (TT3), Free Thyroxine (FT4) and Free Triiodothyronine (FT3) tests, and Thyroid Stimulating Hormone (TSH) measurements, were required to report the IQC information though the national external quality assessment (EQA) network platform. RESULTS: Survey data showed significant variability in all aspects of IQC practice. More than half of the laboratories are using a single concentration level of IQC material and up to 28.6% of the laboratories only use 1(2s) or 1(3s) to monitor IQC results for FT3, TT3, FT4, TT4, and TSH. The medians of the average time intervals of two control tests for FT3, TT3, FT4, TT4, and TSH are 33.6, 35.4, 33.6, 35.4, and 33.6 hours, respectively, more or less 1.4 days. When quality specifications based on biological variation are applied to imprecision evaluation, only 46.3%, 52.1%, 31.3%, 12.8%, and 5.86% laboratories meet the minimum performance for FT3, TT3, FT4, and TT4, 24.5%, 23.6%, 12.8%, and 5.86% of the all meet the desirable performance, and 4.4%, 3.9%, 2.5% and 3.0% of the all meet the optimal performance. While it shows a higher percentage of acceptable laboratories (98.1%, 87.9% and 39.0% meeting the minimum, desirable and optimal performance, respectively) for TSH. CONCLUSIONS: Not-well-designed IQC practices may affect the effectiveness of laboratory IQC and, thus, the adequacy of a laboratory to monitor system performance. Consequently, IQC practice should be designed according to performance of measure method and instrument. Both clinical laboratories and the government should make efforts to improve quality of clinical testing to ensure the patients' safety.

[Effect of enhanced UV-B radiation on photosynthetic structure and photosynthetic characteristics of Mentha piperita].

OBJECTIVE: To reveal the effects of UV-B radiation on the growth of medical plant Mentha piperita, simulate an enhanced UV-B radiation and evaluate intensity of radiation on the photosynthesis of M. piperita. METHOD: Three different levels of UV-B radiation were set in the experiment which included: natural light control (0 W x m(-2)), light UV-B radiation stress (0.15 W x m(-2)) and heavy UV-B radiation stress (0.35 W x m(-2)). The chloroplast ultrastructure, photosynthesis indexes and chlorophyll fluorescence parameters of the M. piperita were observed under the three treatments. RESULT: Although the chloroplast ultrastructure was destroyed to some degree under the light UV-B radiation stress, F(v)/(F)m, F(v)/F(o), qP, phiPS II and ETR could resume to the comparative level of natural light control. At the same time, qN increased firstly and decreased thereafter. But under the high strength UV-B radiation stress, the photosynthetic structures were badly destroyed, which could not recover through protecting mechanism by itself. CONCLUSION: It was showed that M. piperita was able to protect photosynthetic structures by increasing respiration and dissipation when photosynthetic capacity reduced under light UV-B radiation stress. It is demonstrated that M. piperita has high adaptation to light UV-B radiation stress, which is kind of promising medical plant for area with higher UV-B radiation.